BIOLOGY 3200

Principles of Microbiology

LABORATORY MANUAL

Spring, 2005Written by: L. A. Pacarynuk and H. C. Danyk

Revised: January, 2005

TheUniversity ofLethbridge

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TABLE OF CONTENTS

Exercise Page

Biology 3200 Laboratory Schedule 2

Grade Distribution 3

Occupational Health and Safety Guidelines 4

Guidelines for Safety Procedures 5

Exercise 1 – Introduction to Microscopy 7

Exercise 2 – General Laboratory Principles and Biosafety 12

Exercise 3 - Bacterial and Yeast Morphology 14

Exercise 4 – Bacterial Reproduction 20

Exercise 5 – The Ames Test 25

Exercise 6 – Biochemical Tests 28

Exercise 7 – Virology 34

Exercise 8 - Soil and Compost Microbial Ecology 38

Exercise 9 - Applications of Microbiology* 47

Appendix 1 – The Compound Light Microscope 51

Appendix 2 – Preparation of Scientific Drawings 54

Appendix 3 – Aseptic Technique 56

Appendix 4 – The Cultivation of Bacteria 61

Appendix 5 – Bacterial Observation 66

Appendix 6 – Laboratory Reports 67

Appendix 7 – Use of the Spectrophotometer 69

Appendix 8 – Media, Reagents, pH Indicators 71

Appendix 9 – Care and Feeding of the Microscopes 84

*Will require some out of laboratory time for sampling.

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BIOLOGY 3200 LAB SCHEDULESPRING, 2005

Jan. 11 Introduction, MicroscopyJan. 13 General Lab Procedures, Biosafety

Jan. 18 Bacterial MorphologyJan. 20 Bacterial Morphology

Jan. 25 Bacterial MorphologyJan. 27 Bacterial Morphology; Hand in Assignment 1

Feb. 1 Bacterial GrowthFeb. 3 Bacterial Growth - Complete

Feb. 8 Ames Test; Biochemical Tests - Selective and Differential MediaFeb. 10 Ames Test – Complete; Biochemical Tests - Selective and

Differential Media – Complete; Hand in Assignment 2

Feb. 15 Biochemical Tests - IMViC TestsFeb. 17 Biochemical Tests - IMViC Tests – Complete

Feb. 22 Reading WeekFeb. 24 Reading Week

Mar. 1 VirologyMar. 3 Virology

Mar. 8 Virology; Enumeration of Soil and Compost BacteriaMar. 10 Virology – Complete; Enumeration – Complete; Selection of

Unknown and Streak Plate

Mar. 15 Microscopic and Macroscopic Observations of Unknown, WineFermentation

Mar. 17 Microbial Products (using soil/compost samples and unknown),Wine Fermentation

Mar. 22 Evaluation of Microbial Products; Experimental DesignMar. 24 Experimental Design; Wine Fermentation – Complete

Mar. 29 Identification of UnknownMar. 31 Identification of Unknown

Apr. 5 Identification of UnknownApr. 7 Identification of Unknown – Complete; Hand in Lab Report

Thursday Apr. 14 Final Lab Exam (practical)

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Laboratory Grade Distribution:

The laboratory component of Biology 3200 is worth 50% of your course mark. It is distributed asfollows:

• Assignments 7.5%

• Lab Report• Wine Fermentation 15%

• Due Thursday, Apri.l 7 by 4:30 PM• In-Lab Skills Tests 7.5%

• Lab Exam 20%

Performance: Up to 10% of laboratory grade (5 marks out of 50) will be subtracted for poorlaboratory performance. This includes (but is not limited to) failure to be prepared for the

laboratory, missing lab notebook or lab manual, poor time management skills, improperhandling and care of equipment such as microscopes and micropipettors, and unsafe practices

such as not tying hair back, chewing gum, applying lipstick, eating, drinking, or chewing onpencils, and sloppy technique leading to poor results.

Unannounced skills tests will be given during the semester. Students are expected to work

independently on some technical aspect of microbiology and will be graded based on theirtechniques and their results.

As proficiency in microbiological techniques is considered an essential component of the course,

students are only permitted two lab period absences (you do not require any documentation).Missing more than two labs will result in a grade of 0 being assigned for the lab (at this point, it is

recommended that students consult with Arts and Science Advising for the option of completingthe laboratory the following year). Students are still responsible for the material missed (and

their assignments, lab reports etc. will be graded as such). There are no make-up laboratories.

Late Assignments will be penalised as follows: After 4:30 pm but prior to 9:00 am the next day

- -25% (eg. if the assignment is out of 50 points, you will lose 12.5 marks); between 9:00 am and

4:30 pm –50%; etc.

Extensions will only be considered upon application to your lab instructor no less than two days

prior to the due date of the assignment. This application should include documentation and theportion of the assignment completed at that point. Failure to include any evidence of work

completed will result in no extension being granted.

The lab exam (April 14) is comprehensive, covering all aspects of the laboratory. It may contain apractical as well as a theoretical component.

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THE UNIVERSITY OF LETHBRIDGEPolicies and ProceduresOccupational Health and Safety

SUBJECT: CHEMICAL RELEASE PROCEDURE

Precaution must be taken when approaching any chemical release.

1. Unknown/Known Release

• Clear the area• Call Security 2345• Do not let anyone enter the area• Call Utilities at 2600 and request the air be turned off at the release site• Security will immediately notify:

Chemical Release Officer: 331.5201

Occupational Health and Safety: 394.8937394.8716

EMERGENCY CALL LIST 0800 – 1600

2345 SECURITY331-5201 CHEMICAL RELEASE OFFICER

2301 ADMIN. ASSISTANT394.8937394.8716

OCCUPATIONAL HEALTH ANDSAFETY

EMERGENCY CALL LIST 1600 -08002345 SECURITY

331-5201 CHEMICAL RELEASE OFFICER394-8937394-8716

OCCUPATIONAL HEALTH ANDSAFETY

IF THE CHEMICAL RELEASE OFFICER CANNOT BE LOCATED CALL:328-4833 DBS

If the area must be evacuated all employees will be evacuated to the North ParkingLot.

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• GUIDELINES FOR SAFETY PROCEDURES

EMERGENCY NUMBERS

City Emergency 9-911

Campus Emergency 2345Campus Security 2603

Student Health Centre 2484(Emergency - 2483)

THE LABORATORY INSTRUCTOR MUST BE NOTIFIED AS SOON AS POSSIBLE AFTER

THE INCIDENT IF NOT PRESENT AT THE TIME IT OCCURRED.

EMERGENCY EQUIPMENT:

Know the location of the following equipment which will be indicated to you at the beginning ofthe first lab:

1) Closest emergency exit

2) Closest emergency telephone and emergency phone numbers3) Closest fire alarm

4) Fire extinguisher and explanation of use5) Safety showers and explanation of operation

6) Eyewash facilities and explanation of operation.7) First aid kit

GENERAL SAFETY REGULATIONS

1) Eating, drinking or gum chewing is prohibited in the laboratory.

2) Always wash your hands after entering and prior to leaving the laboratory.3) Laboratory coats are required for all laboratories and must be stored in the lab

when not in use.4) Report equipment problems to instructor immediately.

5) Report all spills to the instructor immediately.6) Long hair must be kept restrained to keep from being caught in equipment,

Bunsen burners, chemicals, etc.

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SPILLS

Spill of ACID/BASE/TOXIN: Contact instructor immediately!

BACTERIA SPILLS: If necessary, remove any contaminated clothing. Prevent anyone from

going near the spill. Cover the spill with dilute bleach and leave for 10 minutes before wipingup.

DISPOSAL

Upright Blue Cardboard Boxes:

CLEAN LAB GLASSWEAR - broken glass, Pasteur pipettes, etc. NO CHEMICAL, BIOLOGICAL, OR RADIOACTIVE MATERIALS.

Orange Biohazard Bags:

Petri plates, microfuge tubes, tips, plastic pipettes, etc. All of this material will beautoclaved prior to disposal.

Bacterial Cultures:

Tubes and flasks containing liquid cultures are placed in marked trays for autoclaving.

Bacterial Slides

Used microscope slides are placed into the trays of bleach found at the end of each of thelaboratory benches.

Liquid Chemicals: Place in labelled bottles in fume hood.

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EXERCISE 1

INTRODUCTION TO MICROSCOPY

MICROSCOPY

To view microscopic organisms, their magnification is essential. The microscope is theinstrument used to magnify microscopic images. Its function and some aspects of design are

similar to those of telescopes although the microscope is designed to visualize very smallclose objects while telescopes magnify distant objects.

Magnification is achieved by the refraction of light travelling though lenses, transparent

devices with curved surfaces. In general, the degree of refraction, and hence, magnification,is determined by the degree of curvature. However, rather than using a single, severely-

curved biconvex lens such as that of Leeuwenhoek's simple microscopes, Hooke determinedthat image clarity was improved through the use of a compound microscope, involving two

(or more) separate lenses.

Operation of the Compound Microscope

Students should be familiar with all names and functions of the components of theircompound light microscopes as demonstrated in Appendix 1.

Properties of the Objective Lenses

1. Magnification

Magnification is a measure of how big an object looks to your eye. The number of times that an

object is magnified by the microscope is the product of the magnification of both the objectiveand ocular lenses. The magnification of the individual lenses is engraved on them. Your

microscope is equipped with ocular lenses that magnify the specimen ten times (10X), and fourobjectives which magnify the specimen 4X, 10X, 40X, and 100X. Each lens system magnifies the

object being viewed the same number of times in each dimension as the number engraved on thelens. When using a 10X objective, for instance, the specimen is magnified ten times in each

dimension to give a primary or "aerial" image inside the body tube of the microscope. This imageis then magnified an additional ten times by the ocular to give a virtual image that is 100 times

larger than the object being viewed.

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2. Resolution

Resolution is a measure of how clearly details can be seen and is distinct from magnification. The

resolving power of a lens system is its capacity for separating to the eye two points that are veryclose together. It is dependent upon the quality of the lens system and the wavelength of light

employed in illumination. The white light (a combination of different wavelengths of visiblelight) used as the light source in the lab limits the resolving power of the 100X objective lens to

about 0.25 µm. Objects smaller than 0.25 µm cannot be resolved even if magnification isincreased. Spherical aberration (distortion caused by differential bending of light passing

through different thicknesses of the lens center versus the margin) results from the air gapbetween the specimen and the objective lens. This problem can be eliminated by filling the air

gap with immersion oil, formulated to have a refractive index similar to the glass used for coverslips and the microscope's objective lens. Use of immersion oil with a 100X special oil immersion

objective lens can increase resolution to about 0.18 µm. Resolving power can be increased furtherto 0.17 µm if only the shorter (violet) wavelengths of visible light are used as the light source.

This is the limit of resolution of the light microscope.

The resolving power of each objective lens is described by a number engraved on the objectivecalled the numerical aperture. Numerical aperture (NA) is calculated from physical properties of

the lens and the angles from which light enters and leaves.

Examine the three objective lenses. The NA of the 10X objective lens is 0.25. Which objective lensis capable of the greatest resolving power?

3. Working Distance

The working distance is measured as the distance between the lowest part of the objective lens

and the top of the coverslip when the microscope is focused on a thin preparation. This distanceis related to the individual properties of each objective.

4. Parfocal Objectives

Most microscope objectives when firmly screwed in place are positioned so the microscope

requires only fine adjustments for focusing when the magnification is changed. Objectivesinstalled in this manner are said to be parfocal.

5. Depth of Focus

The vertical distance of a specimen being viewed that remains in focus at any one time is called

the depth of focus or depth of field. It is a different value for each of the objectives. As themicroscope is focused up and down on a specimen, only a thin layer of the specimen is in focus at

one time. To see details in a specimen that is thicker than the depth of focus of a particularobjective you must continuously focus up and down.

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Observing Bacteria

Three fundamental properties of bacteria are size, shape and association.

Bacteria generally occur in three shapes: coccus (round), bacillus (rod-shaped), andspirillum (spiral-shaped). Size of bacterial cells used in these labs varies from 0.5 µm to 1.0µm in width and from 1.0 µm to 5.0 µm in length, although there is a range of sizes which

bacteria demonstrate. Association refers to the organization of the numerous bacterial cellswithin a culture. Cells may occur singly with cells separating after division; showing

random association. Cells may remain together after division for some interval resulting inthe presence of pairs of cells. When cells remain together after more than a single division,

clusters result. Cell divisions in a single plane result in chains of cells. If the plane of celldivision of bacilli is longitudinal, a palisade results, resembling a picket fence. Both bacterial

cell shape and association are usually constant for bacteria and hence, can be used fortaxonomic identification. However, both properties may be influenced by culture condition

and age. Further, some bacteria are quite variable in shape and association and this may alsobe diagnostic.

Micrometry

When studying bacteria or other microorganisms, it is often essential to evaluate the size of the

organism. By tradition, the longest dimension (length) is generally stressed, although width issometimes useful for identification or other study.

Use of an Ocular Micrometer (Figure 1)

An ocular micrometer can be used to measure the size of objects within the field of view.

Unfortunately, the distance between the graduations of the ocular micrometer is an arbitrarymeasurement that only has meaning if the ocular micrometer is calibrated for the objective being

used.

1) Place a micrometer slide on the stage and focus the scale using the 40x objective.2) Turn the eyepiece until the graduations on the ocular scale are parallel with those on the

micrometer slide scale and superimpose the micrometer scale.3) Move the micrometer slide so that the first graduation on each scale coincides.

4) Look for another graduation on the ocular scale that exactly coincides with a graduation onthe micrometer scale.

5) Count the number of graduations on the ocular scale and the number of graduations on themicrometer slide scale between and including the graduations that coincide.

6) Calibrate the ocular divisions for the 40x and the 100x objective lenses. Note that immersion

oil is not necessary for calibration.

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0

0 5

Stage Micrometer (each division = 0.01 mm)

Ocular Micrometer

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Figure 1. Calibration of an ocular micrometer using a stage micrometer. The mark on the

stage micrometer corresponding to 0.06 mm (60 µµµµm) is equal to 5 ocular divisions (o.d.) on the

ocular micrometer. ∴∴∴∴ 1 ocular division equals 60 µµµµm/5 ocular divisions or 12 µµµµm.

Once an ocular micrometer has been calibrated, objects may be measured in ocular divisions andthis number converted to µm using the conversion factor determined.

Bacterial size is generally a highly heritable trait. Consequently, size is a key factor used in

the identification of bacterial taxa. However, for some bacteria, cell size can be modified bynutritional factors such as culture media composition, environmental factors such as

temperature, or other factors such as age.

EXPERIMENTAL OBJECTIVE

In this first exercise, you will calibrate the 40x and 100x objectives of your compoundmicroscope. Then you will use the compound light microscope to assess the shape and

associations of bacteria that have already been fixed to slides and stained. You will also useyour determined calibration factors to evaluate sizes of organisms viewed.

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METHODS:

For each student:

• Compound light microscope• Various prepared slides of bacteria.

• Stage micrometer• Ocular micrometer

• Immersion oil

1) Use the diagram in Figure 1 to calibrate the 40x and the 100x objectives on yourcompound microscopes. Record these values in your lab book as you will then use these

values when measuring cells and structures for the rest of the lab.

Note: Do NOT use immersion oil when calibrating the 100x objective. This is the ONLY

time during the term that you will not use immersion oil with this objective.

2) Use the compound microscope to observe the prepared slides of bacteria using the 10x

and 40x objective lenses. Observe the same slides under the 100x objective usingimmersion oil.

3) Diagram two of the organisms viewed following the instructions found in Appendix 2.

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EXERCISE 2

GENERAL LABORATORY PROCEDURES AND BIOSAFETY

A primary feature of the microbiology laboratory is that living organisms are employed as part ofthe experiment. Most of the microorganisms are harmless; however, whether they are non-pathogenic or pathogenic, the microorganisms are treated with the same respect to assure thatpersonal safety in the laboratory is maintained. Careful attention to technique is essential at alltimes. Care must always be taken to prevent the contamination of the environment from thecultures used in the exercises and to prevent the possibility of the people working in thelaboratory from becoming contaminated. Ensure that you have read over the guidelines onSafety, and those on Aseptic technique (Appendix 3). As well, you should be familiar with thecontents of the University of Lethbridge Biosafety web site:http://www.uleth.ca/fas/bio/safety/biosafety.html

EXPERIMENTAL OBJECTIVES

Students will use fluorescein dye-labelled E. coli cultures to perform a series of exercisesdesigned to illustrate the potential for contamination that is always present when working

with microorganisms. As well, students will become familiar with using aseptic techniquesto handle microorganisms.

METHODS

Benches will be provided with the following:

• Fluorescein-labelled broth culture of E. coli (ATCC strain)(2/bench)• Nutrient agar plates (8/bench)

• Nutrient broth (4 tubes/bench)• Bench coat

• Tape• Gloves

• Hand-held UV lamp• Watch glasses (2/bench)

• Sterile pipettes• Pipette pump

• Tray containing bleach disinfectant

Wear gloves for the entire exercise.1) Tape bench coat onto the bench to cover your working surface.

2) Work individually over the bench coat and prepare a streak plate for single colonies.Label and place in the tray on the side to be incubated.

3) From the same suspension, inoculate one tube of nutrient broth. For steps 4 - 11, work in

pairs.

4) Place a watch glass in the centre of the bench coat.5) Obtain and label 2 NA plates (name, date, organism, distance). Place agar plates on

either side of the glass plate, one 5 cm and the other 10 cm from the watch glass.6) Using a pipette pump, draw up 2 mL of bacteria/fluorescein suspension.

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7) Remove lids from agar plates and set aside.

8) Hold pipette tip 30 cm from glass plate and allow 10 drops to fall (one drop at a time)onto the glass plate. Put any remaining bacterial culture back into the original culture

tube.9) Remove glass plate to disinfectant tray and cover agar plates. Place on a tray on the side

bench.10) Use the hand-held UV lamp in C741 to inspect your bench coat, gloves, and lab coat.

What do you observe?11) Your plates will be incubated for 16-20 hours at 37oC, and then refrigerated at 4oC.

During the next laboratory period, evaluate your plate results and record the number ofcolonies present.

Thought Questions: (Use the Biosafety Web Site as a reference)

• What is an MSDS and where can you find one?

• In Canada, the Laboratory Centre for Disease Control has classified infectious agents into4 Risk Groups using pathogenicity, virulence and mode of transmission (among others)

as criteria. What do these terms mean?• What criteria would characterise an organism classified in Risk Group 1, 2 3 or 4?

Provide an example of an organism found within each group.• There are many “Golden Rules” for Biosafety. Identify 4 common sense practices that

will protect you in your microbiology labs.

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EXERCISE 3

BACTERIAL and YEAST MORPHOLOGY

The Microscopic Examination of Bacteria

Prior to viewing bacteria, two procedures must be performed: 1) fixation and 2) staining.Fixation performs 2 functions: (i) immobilises (kills) the bacteria; and (ii) affixes them to the

slide. The most common fixation procedure for bacteria is heat fixation, whereby the slidecontaining a drop or smear of bacterial culture is passed rapidly once or twice through the

heat of a Bunsen flame.

Staining

Bacteria are almost transparent and hence, unstained bacteria are not readily visible withoutspecial techniques such as phase contrast microscopy (see: Madigan et al, 2003, pp. 56-63) or

dark-field microscopy, which is also referred to as negative staining (Negative staining willbe utilised later on this laboratory). Any procedure that results in the staining of whole cells

or cell parts is referred to as positive staining.

Most positive stains used involve basic dyes where basic means that they owe their colouredproperties to a cation (positively charged molecule). When all that is required is a general

bacterial stain to show morphology, basic stains such as methylene blue or carbol fuchsinresult in the staining of the entire bacterial cell.

Differential stains are used to distinguish bacteria based on certain properties such as cell

wall structure. Differential stains are useful for bacterial identification, contributing toinformation based on bacterial size, shape, and association. Differential staining relies on

biochemical or structural differences between the groups that result in different affinities byvarious chromophores (Appendix 4).

Gram staining behavior relies on differences in cell wall structure and biochemical

composition. Some bacteria when treated with para-rosaniline dyes and iodine retain thestain when subsequently treated with a decolourising agent such as alcohol or acetone.

Other bacteria lose the stain. Based on this property, a contemporary of Pasteur, HansChristian Gram, developed a rapid and extremely useful differential stain, which

subsequently bears his name - the Gram stain used to distinguish two types of bacteria,Gram positive and Gram negative. Gram negative forms, which are those that lose the stain

on decolourisation, can be made visible by using a suitable counterstain. The strength of theGram stain rests on its relatively unambiguous separation of bacterial types into two groups.

However, variables such as culture condition, age or environmental condition, can influenceGram staining of some bacteria.

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The bacterial cell wall is very important for many aspects of bacterial function and hence, the

Gram stain also provides valuable information about the physiological, medicinal and evenecological aspects of the bacteria.

Acid Fast Staining

Members of the genus Mycobacterium contain groups of branched-chain hydroxy lipids called

mycolic acids. Robert Koch first described this property; it allowed him to determine theorganisms present in lesions resulting from tuberculosis. As a result of the presence of these

lipids, these organisms are not readily stained via Gram staining. Instead, cells require heattreatment so that a basic fuchsin and phenol dye penetrate the lipids. Once stained, these

lipids resist decolourisation when treated with acid.

Poly-ββββ-hydroxybutyric Acid (PHB) Staining

PHB granules are common inclusion bodies in bacteria. Monomers of β-hydroxybutyric acid

are connected by ester linkages forming long polymers which aggregate into granules. As

these granules have an affinity for fat-soluble dyes such as Sudan black, they can be stainedand then identified with the light microscope. These granules are storage depots for carbon

and energy.

Endospore Staining

Certain bacteria may produce endospores under unfavourable environmental conditions.Endospores are mainly found in Gram-positive organisms, including the Gram-positive

Clostridium and Bacillus, in the Gram-positive cocci Sporosarcina, and in some of thefilamentous Gram-positive Monosporaceae family. It has also been discovered that Coxiella

burnetii, a small rod found in raw milk that has a variable Gram stain reaction, but a typicalGram-negative cell wall has a sporogenic cycle. When conditions become more favourable,

the endospores will germinate and the bacteria will return to the actively growing anddividing form.

Endospores are highly resistant to heat, chemical disinfectants and to desiccation and

therefore allow the bacterial endospore to survive much more rigorous conditions than thevegetative cells. Endospore resistance is due to several factors, including:

• A decrease in the amount of water compared to vegetative cells• An increase in the amount of dipicolinic acid and calcium ions

• Enzymes which are more resistant to heat• A spore coat which is impermeable to many substances

Endospores may be formed in a central, terminal, or sub-terminal position in the cell and

their shape varies from ellipsoidal to spherical. The location of the endospore in the cell isusually characteristic of the species. For example, the location and shape of the Bacillus

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subtilis endospore is different from the location and shape of the Clostridium endospore.

Therefore, the presence or absence of endospores and the description of the endospore isuseful to a microbiologist as an aid in identification.

The resistant properties of endospores make them difficult to stain, hence heat is used in

conjunction with staining to enable the stain to penetrate into the spore coat.

EXPERIMENTAL OBJECTIVE

The objective of this series of exercises is to perform specialised staining procedures in orderto examine different properties of microorganisms, both bacteria and yeast. These exercises

will also reinforce proper techniques for handling of microorganisms.

METHODS:

For each bench:Stains

• Crystal violet• Safranin

• 5% Malachite green• Carbol fuchsin

• Methylene blue• 20% Sulfuric acid

• Gram’s iodine• Sudan black

• 95% ethanol• Hemo-D (in fume hood)

Equipment

• microbiology kits• compound microscopes

• slides

BacteriaMycobacterium smegmatis

Bacillus thuringiensis

Escherichia coli

Staphylococcus epidermidis

YeastSaccharomyces bayanus

Follow the guidelines for each stain as described below. Work individually.

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Prepare scientific diagrams (Appendix 2) showing results from each stain on separate piecesof paper. These will be collected and graded. If the stain is for a specific structure, ensure

this structure is diagrammed and labelled.

Preparation of Films for Staining – Procedure

• Obtain a clean slide and draw a circle on it approximately 1.5 cm in diameter.

• Turn the slide over.• Flick the tube of culture to mix up the cells, and use a loop to obtain aseptically a

drop of culture. Place this loopful of culture within the circle. Alternatively, if usinga plate culture, first use your loop to add a drop of water to the circle on the slide.

Remove a small quantity of culture and mix with the water to make a smoothsuspension.

• Allow the suspension to air dry. When dry, the film should be only faintly visible; athick opaque film is useless.

• The only fixation required is to pass the slide several times (maximum 10) throughthe bunsen burner flame until the slide is warm but not too hot. If the slide is fixed

until too hot to the touch, the bacteria will be misshapen when observed under themicroscope.

Gram Staining - Procedure

Perform on Bacillus thuringiensis, Escherichia coli, and Staphylococcus epidermidis

1) Prepare smear, dry and heat fix. Flood the smear with crystal violet solution for 1 min.

Gently wash with tap water for 2-3 seconds and remove the water by tapping the slide gentlyon paper towel.

2) Add Gram’s iodine solution to the slide for 1 min. Wash gently with tap water and removeas above.

3) Decolourise with 95% ethanol by dripping ethanol on surface of slide until no more colour isremoved. Rinse gently with water. If too much alcohol is added, the Gram-positive

organisms may become Gram-negative. Remove the water after the last wash.4) Counterstain the slide with safranin for 30 seconds - 1 minute.

5) Wash the slides with tap water, air dry on paper towels, and examine under oil immersion.

Gram positive organisms stain purple; Gram negative organisms, red (pink).

Acid-fast Staining - Procedure

Perform on Mycobacterium smegmatis and on Escherichia coli

1) Flood the dried, heat fixed film with Ziehl’s carbol fuchsin and place on the rack over theboiling water bath.

2) Steam gently for 5 minutes. Do not let the slide dry out. Add more carbol fuchsin asrequired.

3) Wash with tap water to remove excess stain.

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4) Decolourise with 20% sulfuric acid until no more stain comes out. Wash with tap water

to remove excess.5) Counterstain with methylene blue for 1 minute.

Acid fast organisms retain the red stain while others are stained blue.

PHB Staining - Procedure

Perform on Bacillus thuringiensis.

1) Prepare smears of the organism, air dry and heat fix. Flood entire slide with Sudan Black B

and add more stain as the dye solvent evaporates. Stain for at least 10 minutes.2) Pour off excess stain (do not wash) and air dry.

3) Clear slide by dipping in a jar of solvent in the fume hood for 5 sec. Air dry in the fumehood.

4) Counterstain for 1 min. with safranin.5) Wash with water, drain, blot and air dry. Examine with oil immersion objective. Cytoplasm

is pink, lipids are dark grey or black.

Endospore Staining - Procedure

Perform on Bacillus thuringiensis.

1) Prepare smear and heat fix. Cover the dried fixed film with a small piece of paper towel.Saturate this with 5% malachite green.

2) Place the slide on a rack over a boiling water bath. Steam slide for 5-10 minutes in thismanner. Add additional stain as needed - do not allow the slide to dry out during this

procedure. 3) Allow the slide to cool, then rinse with water. Tap over a paper towel to remove excess water

4) Counterstain with safranin for 30 seconds. 5) Rinse slide with water.

6) Allow to air dry, and view.

Endospores will stain green and the rest of the cell pink.

Yeast Staining – Procedure

Perform on Saccharomyces bayanus

1) Prepare a wet mount of the cells using a drop of Methylene Blue.2) Carefully place a cover slip on the cell/stain mixture.

3) View the cells noting size and shape. If you look carefully, you should be able to seebudding cells.

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Thought Questions:

• Why do we stain microorganisms before viewing them with a microscope?

• What is a differential stain? Give two examples of differential stains used in Biology 3200labs.

• Why is immersion oil used to view microscopic organisms?• Gram stains separate microorganisms into two major groups: Gram negative bacteria and

Gram positive bacteria. Describe the differences in the structure of the cell wall of eachtype of bacteria that results in the differential stain result.

• What are endospores? How do they form? Which organisms can produce endospores?• What is the mode of transmission of acid fast organisms? Relate the mode of

transmission to the cell wall structure.

References:

Atlas, R. M. 1997. Principles of Microbiology. Wm. C. Brown Publishers, Toronto.

Madigan, M. T., Martinko, J. M., and Parker, J. 2000. Brock Biology of Microorganisms

Ninth Edition. Prentice-Hall of Canada, Inc., Toronto.

Ross, H. 1992-1993. Microbiology 241 Laboratory Manual. The University of Calgary Press,Calgary.

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EXERCISE 4

BACTERIAL REPRODUCTION

MEASUREMENT OF BACTERIAL GROWTH (See Madigan, et. al., 2003. Chapter 6 Pg.137-

151)

Most bacteria reproduce by an asexual process called binary fission. In this process a single

mother cell produces two identical daughter cells. Cell growth is often equated with increase incell number due to the difficulty in measuring changes in cell size. Under ideal conditions

populations of bacterial cells grow exponentially as cell number doubles at a regular interval orgeneration time (t

d). For example Escherichia coli has a generation time of 20 minutes under

optimal conditions (e.g., 37°C, vigorous aeration and a rich growth medium).

In the laboratory, pure cultures are routinely grown as batch cultures in test tubes andErlenmeyer flasks. A batch culture is prepared by inoculating a fixed amount of liquid medium

with the bacteria then the resulting culture is incubated for an appropriate period of time with nofurther addition of microorganisms or growth substrates.

Cell growth in batch cultures can be divided into four phases. Initially the culture is in a lag

phase where cells are preparing to reproduce. During this time cells are adjusting theirmetabolism to prepare for a new cycle of growth. There is an increase in cell size without

increasing numbers. As cells begin to divide and their growth approaches the maximal rate forthe particular set of incubation conditions established, the culture enters the exponential growth

phase (log phase). One cell gives rise to two, two cells give rise to four, and so on. In this phase,cells are growing and dividing at the maximum growth rate possible for the medium and

incubation conditions. Growth rate is determined by a number of factors, including availablenutrients, temperature, pH, oxygen and other physical parameters as well as genetic

determinants. As nutrients become limiting or waste products accumulate, the growth rate onceagain slows and the culture enters the stationary phase. During this phase, there is no further

net increase in cell number, as growth rate equals the rate of cell death. The final phase of a batchculture is the death phase. During this phase, there is an exponential decline in viable cell

numbers. This decline may be reversed if environmental parameters are modified by theaddition of nutrients, for example.

The rate of growth of bacterial cells is usually monitored by measuring the increase in cell

number. Bacterial cell numbers may be enumerated by a number of methods. Direct count

methods enumerate all cells whether they are viable or not. The most common direct count

method uses a microscope and a specialized counting chamber (e.g., Petroff-Hauser chamber) tocount the number of cells in a known volume of culture. Automated systems such as Coulter

counters may also be used to determine cell number.

In contrast, indirect count methods require the growth of cells in culture in order to enumeratecell numbers. The most common method for enumerating living cells is the viable plate count.

21

Serial dilutions of a cell suspension are prepared and spread on to the surface of a solid agar

medium (spread plate) or incorporated into molten agar that is then poured into sterile petridishes (pour plate). Following a suitable incubation time, the number of colonies growing on and

in the inoculated agar are counted and used to determine the number of viable cells in theoriginal suspension. This method makes the assumption that each colony arose from a single

viable cell or colony forming unit (CFU).

Turbidimetric methods can be used to rapidly assess biomass (e.g., cell numbers). The amount oflight passing through a cell suspension can be determined with a spectrophotometer. The optical

density (OD) is a measure of the amount of light passing through the suspension. A calibrationcurve can be generated using suspensions of known numbers of bacteria.

EXPERIMENTAL OBJECTIVE

In this experiment you will monitor the growth of an E. coli culture by the viable count and

turbidimetric methods. You will determine the number of bacteria (CFU) present in your culturefollowing various time points of incubation. You will establish a growth curve and calibration

curve for OD using the viable count data you collect.

Prelab preparation: Turn on the spectrophotometer and set to 600 nm at least 15 minutes prior

to taking readings.

METHODS

• 100 mL bottles of molten Luria-Bertani (LB) agar• 10% bleach

• Test tube racks• Sterile Petri dishes

• Sterile 5 mL pipettes• Pipette pump

• 10-100 µL micropipettor• 100-1000 µL micropipettor

• Sterile tips for micropipettors• Container of sterile microfuge tubes

• Microfuge tube racks• 65 oC water bath

• Sterile d2H2O• Spectrophotometer blank containing TB broth

• Bacterial waste container• Vortex

• Cuvettes• Spectrophotometer

• Culture flask of E. coli

22

Please work in groups of four. At 20 minute intervals, monitor the growth of your E. coli cultureby determining viable counts as well as optical density following the procedures outlined below.

A. Culture sampling

1) For laboratory sections 1 and 2, each group of four will be assigned a culture flask. Pleasemark the flask with your bench number and lab number. Groups in laboratory sections 3

and 4 will continue to sample from the flask corresponding to your bench. Data from all fourlab sections will be pooled and posted on the Biology 3200 web site.

2) Everyone in the laboratory will be sampling at the same time. Samples will be collected threetimes at 20 minute intervals. For labs 1 and 2, these correspond to: 9:45 am, 10:05 am, 10:25

am, and for labs 3 and 4: 11:10 am, 11:30 am, and 11:50 am. Your laboratory instructor willset a timer so that everyone is coordinated. Prior to beginning, designate two individuals in

your group to be responsible for obtaining optical density (OD) readings at each time point.The other two individuals will prepare and plate appropriate serial dilutions for viable

counts.3) At 20 minute intervals aseptically obtain one 5 mL sample of culture and immediately place it

in a spectrophotometer tube. This material will be used to measure optical density (OD)(Section B). After reading, dispose of your 5 mL sample of culture in the waste beaker

provided. Rinse the spectrophotometer tube using the squirt bottle of bleach provided andthen dispense the solution into the waste beaker.

4) Remove another 100 µL of the culture and place it into a sterile microfuge tube. Label thistube Tube 1. Use this culture for Section C.

B. Determination of optical density (please read Appendix 7)

1) Zero the spectrophotometer as outlined in Appendix 7.

2) Place the spectrophotometer tube containing your culture into the spectrophotometerand record the optical density (Absorbance) reading in your lab book and in the table on

the blackboard. If the reading is greater than 0.7, you must dilute your sample andremeasure the optical density. It is suggested that you begin by diluting your sample 1:1

with the TB provided. Make note of the dilution that you prepare in order to obtain anaccurate absorbance reading. Multiply the absorbance by the dilution factor to obtain the

final reading.

23

C. Enumeration of viable bacteria

1) Remove four sterile microfuge tubes from the container on the side bench. In order thatyou don’t contaminate all of the tubes, gently tap out four tubes from the container rather

than using your hand to grab tubes.2) Set up your serial dilutions according to the information in Table 4.1. Aseptically pipette

900 µL of TB into Tube 1 that already contains 100 µL of bacterial culture. You have nowcreated a 1:10 dilution. Mix well using the vortex mixer. Create the remaining serial

dilutions (tubes 2-4) in the same manner. Use fresh tips for each transfer.

Table 4.1. Preparation of serial dilutions from E. coli culture sampled at 20 minute

intervals

Tube

Number

Amount of

sterile TB (µµµµL)

Amount of

Culture

Final Dilution

Factor

1 900 100 µL fromculture flask

10-1

2 990 10 µL from tube1

10-3

3 990 10 µL from tube2

10-5

4 900 100 µL fromtube 3

10-6

5 (for labs3 and 4

only)

900 100 µL fromtube 4

10-7

The dilution sequence will be set up each time you take a sample from your culture flask.

3) Labs 1 and 2 will be plating the contents of Tube 3 and Tube 4 (10-5 and 10-6 dilutions).Labs 3 and 4 will be plating the contents of Tube 4 and Tube 5 (10-6 and 10-7). Obtain 2

sterile Petri dishes. Label the bottom (not the lid) of the plate with the time the samplewas taken, your group name, and the dilution. 20 mL corresponds to where the bottom

edge of the lid is when the lid is on the Petri dish.4) Add the contents of Tube 3 to the appropriately labelled sterile Petri dish. Obtain a bottle

of molten LB agar from the water bath at the side of the lab, and add approximately 20mL of molten agar (after flaming the mouth of the bottle) to the diluted culture. Swirl

carefully to mix the inoculum evenly with the medium. Label the bottle of molten agarwith your group name and replace it immediately in the water bath.

5) Follow the instructions provided in step 4 above to plate out the contents of Tube 4.

24

6) When the agar has solidified, place the inverted plates on a tray at the side of the lab.

The plates will be incubated for 16 – 20 hours at 37°C and refrigerated until the next labsession.

The next laboratory period:

7) Examine the plates carefully and select the plate where the bacterial count rangesbetween 30 and 300 colonies.

8) Record the number of colonies on the plates in your lab notebook and in the chart on theboard. Complete data sets will be available on the Biology 3200 web site.

9) Use class data to determine the average number of bacteria per mL of culture.10) Prepare graphs from class data comparing i) OD vs time (on semi-log graph paper); 2)

CFU/mL vs time (on semi-log graph paper); 3) OD vs CFU/mL (on arithmetic graphpaper). The first two graphs are growth curves; the third graph is a standard curve

allowing for correlation between OD and CFU/mL (Please see Madigan, et. al, 2003)

Prepare a Results and Discussion section upon conclusion of this laboratory according to theinformation found in Appendix 6.

Thought Questions:

• Use your graph(s) to calculate generation time of E. coli.

• Compare your value to that from the literature. Do the values differ? Why might this be?• Compare and contrast indirect and direct methods of counting bacteria.

• Use your standard calibration curve to calculate the CFU/mL of culture for an undilutedsample in which the OD was 0.75.

• Based on the differences in ingredients, what are the differences between growing cells on LBversus TB? Why is TB used for generating growth curves of E. coli rather than LB?

25

EXERCISE 5THE AMES TEST

MUTATION AND RECOMBINATION (See Madigan, et. al., 2003. Chapter 106 Pg. 265-276)

You have learned about some of the advantages of using a model system in your study of theeffect of UV light on DNA in Biology 2000 (Introduction to Genetics). The Ames test also makes

use of a model system in order to measure the mutagenic potential of compounds. This test is areversion mutagenesis assay and uses strains of the bacterium Salmonella that have point

mutations in various genes in the histidine operon. These His- mutants are unable to synthesisehistidine and therefore unable to grow on minimal media lacking histidine. When the His- tester

cells are cultured on a minimal agar medium containing trace amounts of histidine, a small andrelatively constant number of cells per plate spontaneously revert to His+ and subsequently

reproduce and form colonies. Incorporation of a mutagen into the agar increases the number ofrevertant colonies per plate, usually in a dose dependent manner.

EXPERIMENTAL OBJECTIVE

You will make use of the Ames test in order to evaluate the mutagenicity of a selection ofcompounds.

PRE-LAB PREPARATION

Each class should bring in a total of three household compounds they would like to test. These

will be decided in advance. Note that these compounds must be known (ie “mystery liquid”from the garage is not acceptable) and they must be taken home again once Period 1 of the lab is

finished.

METHODS:

For each lab:• 100 mg/mL Sodium Azide (CAUTION: MUTAGEN!)

• Ethidium bromide (10 mg/mL)• Micro Kits

• Gloves• Sterile water

• 3x Liquid cultures of Salmonella strains 1535 and 1538 in NB supplemented with NaCl• Top agar overlay in 50oC water bath (2 mL per tube)

• Test tube with 2 mL mark indicated (at pouring station)• Minimal salts plates (15 per lab)

• Vortex mixer (at pouring station)• Bunsen burner (at pouring station)

• Test tube racks• Sterile filter paper disks

26

• Forceps

• 3x micropipettors (10 – 100 µL)• Sterile tips

• 5x beakers with biohazard bags• Small vials containing 95% ethanol for flaming

Set up your experiment as follows in the Table:

Compound to be TestedBench #

Water Unknown1

Unknown2

Unknown3

SodiumAzide

Ethidiumbromide

+ 1535 +15351+ 1538

+ 1538 +15352+1538

+15353+1538

+15354+1538

+15355+1538

1) For each plate, you will be creating an overlay using a single strain mixed with the top

agar. The top agar has had a trace amount of histidine and biotin added. Using theTable as a guide, obtain and label the appropriate number of minimal salts plates.

Why is it necessary to add a trace amount of histidine to the top agar?

2) Have your plates labelled, and take to the station set up at the back bench. Set a

micropipettor to 50 µL. Remove one tube of agar overlay from the waterbath, andaseptically add 50 µL of liquid culture to the tube. Vortex to mix and pour over the

surface of your agar plate. Clean up your work surface prior to going back to yourbench.

Note: you must work very quickly in order to avoid the top agar solidifying.

3) Allow your agar to solidify for 10 minutes.

Wear gloves for any handling of the potential mutagens!

4) Flame forceps to sterilise. Note that this does not mean holding forceps in the flame of

your Bunsen burner until redhot! Rather, dip the forceps in ethanol, and wave throughthe flame. Allow the ethanol to burn off. Pick up a sterile filter paper disk and dip in the

appropriate mutagen. For the cigarette extract, you will need to go to the fume hood todo this.

27

7) Tap the filter paper several times to remove excess liquid. Hold the filter paper for a few

moments to ensure that liquid doesn’t drip all over your plates. Place the filter paper inthe centre of the plate with the solidified overlay. Tap gently to ensure that the filter

paper stays in place.

8) Incubate your plates for 48 hours at 37 oC. In the next lab, enumerate the number ofcolonies on each plate and record the results on the board.

Thought Questions:

• What specific mutations in the His operon do each of the Salmonella strains usedcontain?

• Evaluate the compounds tested for mutagenicity. What kind of mutations arebeing caused by the compounds tested? (use the information from the first

Thought Question to answer this)• Typically, mutagens are first mixed with liver extract prior to carrying out the

Ames test. What would be the purpose of this step?

References:

Ames, B.N., Durston, W.E., Yamasaki, E., and Lee, F.E. 1973. Carcinogens are mutagens: a

simple test combining liver homogenates for activation and bacteria for detection. Proc.Natl. Acad. Sci. U.S.A. 70:2281-2285.

Ames, B.N., Lee, F.E., and Durston, W.E. 1973. An improved bacterial test system for the

detection and classification of mutagens and carcinogens. Proc. Natl. Acad. Sci. U.S.A.70:782-786.

Ames, B.N., McCann, J., and Yamasaki, E. 1975. Methods for detecting carcinogens and

mutagens with the Salmonella-microsome mutagenicity test. Mutational Research 31:347-364.

Madigan, M. T., Martinko, J. M., and Parker, J. 2003. Brock Biology of Microorganisms

Tenth Edition. Prentice-Hall of Canada, Inc., Toronto.

28

EXERCISE 6

BIOCHEMICAL TESTS (Selective and Differential Media; IMViC Tests)

Normally, the coliform group of bacteria is used to indicate the pollution of water with fecalwastes of humans and animals, and thus, the suitability of a particular water supply for

domestic use. The term coliform is used to describe aerobic and facultatively anaerobic Gramnegative rods that ferment lactose with gas formation. Most, but not all organisms within

this group are intestinal in origin; for instance, Escherichia coli. Consequently, presence oflactose fermentors in a sample of water provides circumstantial evidence of pollution by fecal

wastes, and may suggest the presence of pathogenic bacteria such as members of the generaSalmonella and Shigella. These pathogens, in addition to non-pathogens such as E. coli are

members of the Enterobacteriaceae family. In order to identify the organisms present in thewater, several biochemical tests that rely on differences in the chemical composition of media

used may be performed (see Appendix 4 and Appendix 8 for more details).

SELECTIVE AND DIFFERENTIAL MEDIA:

I. Media for Isolation of Enterobacteriaceae

A strategy for bacterial isolation involves the use of selective media, media with specificcomponents that promote the growth of some bacteria and inhibit the growth of others.

Selectivity may be achieved in three ways:• by adding something to the medium to discourage the growth of species not

required• by altering the pH of the medium

• by omission of some ingredient required by most bacteria, but not by the organism tobe isolated

Differential media contain specific biochemical indicators that demonstrate the presence of

certain substances characteristic of certain bacteria. Thus, differential media are useful forbacterial identification.

Eosin Methylene Blue Agar (EMB Agar)

EMB is both a differential and selective plating medium recommended for use in the isolationof Gram-negative bacilli and the differentiation of lactose fermentors from non-lactose

fermentors.

EMB agar contains the two indicators, eosin Y and methylene blue as well as thecarbohydrate lactose. Eosin (an acidic dye) reacts with methylene blue (a basic stain) to form

a compound of either acidic or neutral nature. The acid produced by lactose fermentors issufficient to cause this dye compound to be taken up by the cells. Non-lactose fermentors are

colourless because the eosin and methylene blue compound cannot be taken up by the cells.The basic stain methylene blue inhibits bacterial growth, particularly that of Gram positive

29

bacteria (due to their cell wall composition). Eosin methylene blue (EMB) agar is thus

selective for Gram negative bacteria.

MacConkey Agar

MacConkey agar is a differential and selective plating medium recommended for use in the

isolation of Gram-negative bacilli and the differentiation of lactose fermentors from non-lactose fermentors. The differential action of the MacConkey agar is indicated by the colonies

of coliform bacteria becoming “brick red” in colour. This occurs when the coliforms utilisethe lactose producing acids. The decrease in pH results in the uptake of the indicator neutral

red by the cells. Non-lactose fermentors are colourless and transparent. Production of acidmay also result in a zone of precipitated bile surrounding the colony. Bile salts and crystal

violet present in the medium inhibit Gram-positive bacteria from growing.

II. Acid Production from Carbohydrates

As demonstrated with MacConkey Agar, bacteria vary in their ability to ferment varioussugars. Products of fermentation are often acids and hence, pH changes can demonstrate

successful fermentation. In addition, gas (usually but not always CO2) is often producedduring fermentation, offering another indicator.

Hugh and Leifson’s method for demonstrating the presence of the products of fermentation

consists of a semi-solid medium containing peptone (short chains of amino acids), thecarbohydrate of interest (usually glucose or lactose), and a pH indicator, Bromothymol blue.

Tubes are stab-inoculated all the way to the bottom of the tube, so as not to introduce oxygeninto the medium. Several reactions may be observed. Facultative organisms will produce an

acid reaction (the indicator changes to yellow) throughout the entire tube of medium. Theacid reaction produced by oxidative organisms is apparent first at the surface, extending

gradually downwards into the medium. Note that organisms that oxidise glucose aregenerally unable to ferment any carbohydrate. Strict fermentors will produce an acid

reaction at the bottom of the tube.

Organisms unable to use the carbohydrate may be able to grow using the peptone in themedium. Production of alkaline products result in the formation of a blue colour at the top of

the tube (although this does not indicate that the organism is aerobic).

III. Motility Medium

This medium contains triphenyl tetrazolium chloride and a small concentration of agar inorder to make the medium semi-solid. TTC is reduced when broken down by the organism,

and the TTC turns red where this has occurred. If the organism is facultative and motile, itmoves throughout the entire tube of medium and the whole tube becomes red. If the

organism is aerobic and motile, the top of the tube becomes red.

30

METHODS

For each bench:

• 3 plates each of MacConkey and EMB media• 5 known broth cultures

• 1 ‘unknown’ broth culture• 6 tubes of Hugh and Leifson’s (H & L) lactose medium

• 6 tubes each of motility mediumPlease work in groups of four.

1) Divide your three MacConkey and three EMB plates in half and streak inoculate themwith the six bacterial species provided. After incubation at 37°C for 48 hours, observe,

and describe the various cultures on the plates in your lab book. Generate a table ofresults summarising growth and properties of all bacteria on the two media.

2) Determine the lactose fermentation ability of all of the bacteria provided. These tubes areinoculated using a stab technique. Use the probe to aseptically remove a small amount of

bacterial culture, and then stab the probe to the bottom of the tube of medium withoutmixing the medium around. Inoculate each tube with one of the bacterial species and

label appropriately. Tubes will be incubated for 48h at 37°C. After incubation, observetubes and record results in your lab book.

3) Work collectively to inoculate your motility medium tubes. Again, as this medium issemi-solid (the stab technique is used for all semi-solid media in this course), use a probe

and stab the culture down to the bottom of the tube and remove the probe. Do not mixthe probe around in the tube. Tubes will be incubated for 48 h at 37oC. After incubation,

observe tubes and record the results in your lab book.

IMViC TESTS

Only preliminary taxonomic assessment of bacteria can be made on the basis of microscopicsize, shape, association, and Gram staining. Information regarding natural occurrence is also

valuable since bacteria generally occur in specific habitats. This is particularly the case forfastidious bacteria, those with very specific nutritional and environmental requirements.

However, even when supplemented with habitat information, bacterial identification basedon microscopic assessment is generally incomplete.

Confident bacterial identification can be made based on biochemical tests, and for certain

pathogens, or for examining microbial presence in specific environments, series of diagnostictests have been developed. For example, the IMViC tests are used routinely to confirm the

presence of coliform organisms in water. “IMViC” is an acronym for ‘Indole, Methyl Red,Voges-Proskauer, and Citrate utilisation’ tests (the “i” is inserted for ease of pronunciation).

I. Indole Formation - Utilisation of Tryptophan

31

When cultured on peptone water, a liquid medium containing tryptophan, certain bacteria

will produce indole. The presence of this indole is readily revealed through addition ofKovak's reagent, producing a pink colour. This reagent contains the organic solvent amyl

alcohol that extracts the coloured (pink) substance.

II. The Methyl Red (MR) Test - Mixed Acid Fermentation Pathway

Fermentation of glucose via the mixed acid fermentation pathway results in the formation ofa number of organic acids such as lactic and acetic acid. If this is a primary fermentation

pathway of a bacterium, a noticeable drop in pH will occur with incubation on MRVP media.This decrease in pH can be revealed by a methyl red solution which is yellow under neutral

conditions and red at a pH less than 5.

III. The Voges-Proskauer (VP) Test - The Butanediol Fermentation Pathway

An alternate fermentation pathway performed by some other bacteria results in theformation of a non-acidic product, butanediol and hence, is named for this product. The

occurrence of the pathway may be determined by a biochemical test for an intermediatecompound in the pathway, acetoin (acetyl methyl carbinol), which is detected by the Voges-

Proskauer test.

IV. Citrate Utilisation - Growth Using A Single Carbon Source

The nutritional requirements of different bacteria vary considerably and these can provideuseful information contributing to biochemical identification. In Simmon's citrate agar,

citrate, in the form of sodium citrate, is the sole carbon source. Organisms able to utilise thecitrate grow on the surface of the medium and due to oxidative formation of sodium

carbonate, raises the pH of the medium changing it from green to blue (bromothymol blue isthe indicator).

V. Urea Hydrolysis

Some bacteria can produce urease, an enzyme which hydrolyses urea into ammonium and

carbon dioxide. The presence of this enzyme is detected by growing the bacteria in amedium containing urea and a pH indicator, phenol red. If ammonium is produced as a

result of urea hydrolysis, the increase in pH will turn the medium to a violet-red colour.

32

METHODS:

For each bench:• 6 broth cultures, one of which is an ‘Unknown’

• 6 MRVP broth tubes• 6 indole broth tubes

• 6 Simmons citrate agar slants,• 6 Urea broth tubes

Please work in groups of four.

1) Inoculate 6 Indole broth tubes separately with the 6 bacteria. After 48h of incubation at

37 oC, add 20 drops (1 mL) of Kovak's reagent (work in the fume hood and leave your

tubes in the fume hood to develop and observe). Shake and look for the formation of a

pink colour in the top (organic) phase; it may take 20 minutes to develop. The pinkcolour is a positive result, indicating the ability to use tryptophan.

Note, please place tubes containing amyl alcohol in a separate rack in the fume hood as

this material needs to be disposed of separately.

2) A single culture solution (peptone, glucose, potassium phosphate) will be used for boththe methyl red and Voges-Proskauer tests. Inoculate 6 MRVP tubes with the 6 bacteria

provided, one culture into each tube.

• After 48h of incubation at 37°C, remove about 1/4 of the broth (=2 mL = 40 drops)from the MRVP tube and transfer that to another test tube. Add 3-5 drops of methyl

red solution. An immediate red reaction provides a positive response to the test,indicating the presence of mixed acid fermentation. A yellow or orange colour

represents a negative response.

• As the same solutions are used for the MR and VP, remove an additional 2 ml ofculture solution and add 1 ml α-napthol (Barritt's reagent A - 1 ml is about 20 drops)

and 1 ml 40% KOH (Barritt’s reagent B; caution - this is caustic). Shake vigorously for30 seconds.

• Shake the tubes frequently and observe for up to 30 minutes for the formation of a

red colour that represents a positive VP test. A yellow or brown colour is a negativeresult.

3) Inoculate 6 Simmon's citrate agar slants separately with the 6 bacteria. For these

inoculations, use your loop to smear cells along the surface of the slant. Incubate tubesfor 48 h at 37°C.

33

• After incubation, observe colours on the surface and down through the tubes. A darkblue colour is a positive result while green indicates a negative test for citrate

utilisation.

4) Inoculate 6 urea slants separately with the 6 bacteria. After incubation for 48h at 37°C,observe for the development of a violet-red colour.

5) After completing the Indole, MR, VP, Citrate and Urea tests, collaborate with the other

students at your bench to generate in your lab book tables of results for all bacteria in alltests.

Thought Questions:

• Identify your unknown. Can it be any of the knowns? Why or why not? Provide

evidence to support your choice of organisms.• Compare and contrast chemically defined and complex media. Provide two examples

of complex media used in this exercise and explain why these media are consideredcomplex.

• Provide two examples of compounds responsible for buffering in media.• Is agar a nutritionally complete substrate for microbes? Why or why not?

• Design a defined medium for an organism that can grow aerobically on acetate as acarbon and energy source.

• In this laboratory, would you classify the organisms used as photoautotrophs,photoheterotrophs, chemoautotrophs, or chemoheterotrophs? Explain your choice(s).

34

EXERCISE 7

VIROLOGY

(Please review the material on sewage treatment posted on the Biology 3200 web page)

EXPERIMENTAL OBJECTIVE

The objectives of this series of exercises are first to isolate coliphage from filtered raw and treated

sewage obtained from the Lethbridge Wastewater Treatment Plant, to examine the plaque

morphologies, and to prepare phage isolate from one particular plaque. Using this phage isolate, the

phage titre will be determined, and the host specificity of the phage will be examined using several

enteric bacterial strains. These exercises will demonstrate standard techniques in phage isolation and

manipulation.

Prior to the laboratory, sewage samples were collected at the areas indicated on the schematic posted

on the web page. Both samples were stored at 4 oC prior to filtering, for up to 1 week. On the morning

of the lab, samples were filtered twice using 0.45 µm filters.

PART A - ISOLATION

METHODS:

For each bench:

• Luria Methylene Blue agar plates

• Overnight culture of Escherichia coli K12

• Bottle of molten Luria agar overlay (at 60 oC)

• Sterile test tubes

• Test tube rack

• Micropipettor (100 µL – 1000 µL)

• Sterile tips

• Microbiology kits

For the lab:

• Vortex mixer

• Water bath set to 60 oC

• Raw and treated sewage filtrate

• Test tube showing 4 mL mark

Work in groups of 4.

Note that sewage filtrate contains human pathogens. Work very carefully. Students who are

clearly unprepared or are sloppy will be asked to leave the lab.

Procedure

1) Obtain a tube of culture of E. coli K12.

35

2) Obtain 5 Luria Methylene Blue agar plates, and 5 sterile test tubes. Label your 5 tubes according

to Table 7.1.

Table 7.1 Experimental set-up for isolation of coliphage from sewage.

Contents (µµµµL)Tube #

K12 Raw Sewage

Filtrate

Treated Sewage

Filtrate

1 500 0 0

2 0 500 0

3 0 0 500

4 500 500 0

5 500 0 500

3) Pipette the appropriate amount of filtrate and/or cells into each of your labeled test tubes. Leave

the tubes at room temperature on your bench to incubate for 20 minutes to allow the phage to

adsorb to the cells.

4) While your cultures are incubating, label your Luria Methylene Blue plates according to Table 7.1.

Mark the level of 4 mL on each of your tubes using the marked test tube on the side bench as a

guide.

5) Starting with Tube 1, aseptically pour molten agar into the tube up to the level of 4 mL. Vortex to

mix, then immediately pour the contents over the surface of the appropriately labeled plate. Swirl

the plate gently to ensure that the entire surface is covered with the agar.

6) Repeat step 5 for the remaining tubes and plates.

7) After 10 minutes, the overlay should be set. Invert your plates and place them on a tray on the

side bench to be incubated. Plates will be incubated at 37 oC for 16 – 20 hours, then stored at 4 oC

until the next laboratory period.

The next laboratory:

Work in groups of four.

MATERIALS

• Pasteur pipettes

• Bulbs

• Chloroform (in the fume hood)

• Vortex mixer

• Phage dilution buffer

• Plates from last lab

• 1 dissecting microscope per bench

36

• Microfuge tubes (sterile)

• 1 mL pipettes and propipettors

• Microfuge racks

• Labeled microfuge rack on the side bench for class tubes

5) Obtain your plates. Examine them carefully. Record the number of plaques present for both raw

and treated filtrate. Is there any difference?

6) Make detailed observations of plaque morphology. Features to look for include size, shape, and

turbidity (clear vs cloudy). Use the dissecting microscopes for your observations.

7) After making observations, obtain a microfuge tube and aseptically add 1 mL of phage dilution

buffer to your tube. Label with your group designation.

8) Use a Pasteur pipette (with a rubber bulb attached) to remove a plaque (squeeze the bulb, insert

pipette into the agar over a plaque, gently release bulb to remove a plug of agar containing the

plaque). Note that for each group of 4, two morphologically distinct plaques should be chosen.

Release plaque into the prepared tube of phage dilution buffer.

9) Vortex vigourously to disperse the agar.

10) Move to the fume hood and use a Pasteur pipette to add a drop of chloroform to your tube. Vortex

the mixture once again.

What does the chloroform do?

Place your tubes in the rack on the side bench. The tubes will be stored at 4 oC allowing the phage to

elute from the agar into the buffer.

PART B – HOST RANGE

METHODS

Overnight cultures of:

• Salmonella typhimurium strain 1535

• E. coli strains CSH121 and CSH125 and K12

• Proteus vulgaris

• Enterobacter

Other supplies:

• Phage dilution buffer

• Micropipettors and sterile tips

• Autoclave waste disposal

• Luria Methylene Blue agar plates

• LB plates

• Bottle of molten Luria agar overlay (at 60 oC)

• Sterile test tubes

• Test tube indicating 4 mL mark

• Test tube rack

• Micropipettor (100 µL – 1000 µL)

• Sterile tips

37

• Microbiology kits

For determining phage titre:

1) Prepare serial dilutions of your phage in dilution buffer (10-2, 10-4, 10-6, 10-8) in microfuge tubes.

Vortex each tube as you create each dilution. Ensure that you use fresh tips for each transfer.

2) In separate, labeled sterile test tubes, mix 500 µL of each dilution with 500 µL of host strain E.

coli K12. Sit for 20 minutes of incubation time at room temperature. Mark the 4 mL mark on

each test tube while mixtures are incubating.

3) Plate your mixtures as per Part A of this exercise.

4) The next day, count plaques and determine the titre of your phage.

For determining host range:

1) Prepare spread plates on LB for each organism to be tested. (use the instructions found in

Appendix 3, although this should be a review from previous courses!). Label each plate clearly.

Use 100 µL of liquid culture to create a uniform lawn.

2) When lawns are dry, divide plates into four quadrants. In each quadrant, spot 20 µL of each phage

dilution. Do not invert. Plates will be incubated at 37 oC overnight.

3) The next day, score as + or – for phage growth on each host.

Thought Questions:

• Based on the schematic found on Dr. Brent Selinger’s web site, what step(s) is/are most likely

responsible for the difference in coliphage numbers between raw and treated sewage?

• Have you isolated more than one type of phage? How might you be able to tell?

• To what components of the bacterial cell to phage typically adhere?

38

EXERCISE 8

SOIL AND COMPOST MICROBIAL ECOLOGY

Soil Bacteria

The microflora of the soil exist as a complex food chain that brings about the release of

nutrients from dead plant material on the surface. The surface layer of newly fallen plantmaterial is called the litter and chemically it is composed of insoluble materials such as

cellulose, hemicellulose and lignin. Only a few organisms, usually fungi, are able to utilisethese high molecular weight compounds since carbohydrates, amino acids, vitamins and

other growth factors are lacking. However, once microorganisms do begin to decompose thelitter, the chemical structure of the litter is modified and the organisms produce end-products

that are released into the environment and become available for use by others. Death of theseorganisms also provides new small molecular weight compounds that may then be utilised.

Bacteria are able to utilise the end-products of fungal metabolism. Nematodes and protozoafeed on the bacteria and mites and other animals live on the nematodes and protozoa. In this

way nutrients are recycled.

Compost Bacteria

Composting is a microbial process whereby plant matter including lignin is partially

converted to humus, therefore supplementing the organic content of soil. The process isinitiated by mesophilic heterotrophs and initially is characterised by a temperature increase

up to 55 – 60oC for a few days where thermophiles such as Bacillus stearothermophilus andThermomonospora are active. The temperature then decreases, followed by several months of

curing at mesophilic temperatures, where again, mesophiles predominate. Composting isnot exclusively carried out by bacteria; fungi such as Aspergillus fumigatus, and Geotrichum

candidum, are also involved.

EXPERIMENTAL OBJECTIVES

In this experiment, you will prepare serial dilutions of compost and of soil samples and plateout the appropriate dilutions. After incubation, you will determine the number of bacteria

isolated in your two samples, and assess the microorganisms for their ability to utilisecarboxymethylcellulose, casein, starch and xylan. You will choose one organism from either

soil or compost, use biochemical tests to identify the microorganism you have chosen, anduse the class results to compare and contrast microbial diversity in soil and in compost.

39

METHODS:

• Microbiology kits

• Soil• Compost

• Balance• Bottles containing 100 mL sterile water

• Vortex• Petri dishes

• 250 mL bottles of molten peptone yeast extract agar in 60oC water bath• 9 mL water blanks

• Sterile pipettes, propipettors• Crystal violet

• Safranin• Carbol fuchsin

• Gram’s iodine• PYE broths

• Sudan black• 95% ethanol

Out of your group of four, one pair will prepare enumeration plates for soil, while the other

pair will prepare enumeration plates for compost.

A. Enumeration of bacteria in soil and in compost

1) Weigh 1 g of soil or of compost provided and add to 100 mL of sterile distilled water.This is dilution #1 (1:100). Shake the suspension for 5 minutes.

For those pairs working with soil, please follow the instructions outlined in steps 2-5; those

pairs working with compost, please follow steps 6-9.

2) Use the sterile 9 mL distilled water blanks provided to create serial dilutions of your

soil. Please ensure that you vortex your samples well prior to making each newdilution. You will require 10-4 and 10-5 dilutions of soil.

3) Add 1 mL of the 10 -4 dilution to each of two sterile, labeled Petri dishes and 1 mL ofthe 10 -5 dilution to two labeled Petri dishes.

4) Obtain a 250 mL bottle of molten peptone yeast extract agar – label with tape and leave in

waterbath when not in use.

5) Use the sterile 9 mL distilled water blanks to create serial dilutions of your compost. Please

ensure that you vortex your samples well prior to making each new dilution. You will require

10-4, 10-5, and 10-6 dilutions of your compost.

40

6) Add 1 mL of the 10 -4 dilution to each of two sterile Petri dishes, 1 mL of the 10 -5

dilution to two sterile Petri dishes, and 1 mL of the 10-6 dilution to two sterile Petri

dishes.

7) Obtain your labeled 250 mL bottle of molten peptone yeast extract agar from thewater bath.

8) Add approximately 20 mL of medium to the plates prepared in Step 7. Swirlcarefully to mix the inoculum evenly with the medium.

Both pairs should make note of step 9:

9) The plates will be incubated for 24 hours at 30oC and refrigerated until the next labsession.

Results (please work as a group of 4 to enumerate the organisms)

Examine both sets of plates carefully and select the plates where the bacterial count ranges

between 30 and 300 colonies. Record the number of colonies on the plates in your notebooksand on the board and determine the mean (± standard deviation) number of bacteria per g of

soil and of compost.

B. Isolation and characterization of bacteria from soil or compost

Two classes will identify organisms from compost bacteria plates while the remaining classeswill identify organisms from soil bacteria plates. Your instructor will indicate what plates to

remove your unknown from.

Work individually to complete Part B of this exercise.

Choose a morphologically distinct colony from the plate provided by your instructor andprepare a streak plate for single colonies on peptone yeast extract agar (Appendix 3).

The plates will be incubated for 24 hours at 30oC and then stored at 4oC.

Prepare a Gram stain of the pure culture and record the cell morphology. Record the colony

morphology of the culture (see Appendix 6). Prepare a liquid culture of a single colony usingPYE broth. Use this culture to inoculate all of the biochemical test media you use.

Use the dichotomous key provided to develop a detailed outline of the series of steps you

plan to take to identify your unknown. This outline should include tests to carry out as wellas dates when you intend to do these tests. This outline must be handed in and approved

before you will be allowed to proceed.

The following media and reagents will be available for you to utilise as you attempt toidentify your unknown:

41

• nutrient agar• endospore stain reagents

• capsule stain reagents• hydrogen peroxide (catalase test)

• oxidase reagent (oxidase test)• IMViC reagents

• indole broths• MRVP broths

• citrate slants• urea broths

• litmus milk broths• mannitol broth (with phenol red indicator)

• H & L medium containing glucose• H & L medium containing lactose

• sucrose agar• motility medium (with TTC)

42

1. Gram stainGram positive ......................................................................................…………………...... 2Gram negative...........................................................................................………………….16

2. Cell morphologybacillus or spirillus……...................................................................………….........………...3coccus….................................................................................................……………………..12ovoid….....................................................................................……………………Azotobacter

3. Endospore stainpositive.......................................................................................................……..……………..4negative ...................................................................................…………………................... 6

4. 1 True endospores......................................................................................…………………........... 52 Special spore types..........................................................................................……………………6

5. Aerobe or facultative anaerobe .................................................................………………..BacillusObligate anaerobe..............................................................................…………………..Clostridium

6. Bacillus cells may be branched, no true mycelium................….....……………........................ 7Bacilli form true mycelium...............................................................................………………….10

7. Cells pleomorphic depending on age of culture...................................................……………...8Cells bacillus-shaped only. Club-shaped swelling may be present

in young cultures....................................…................................................…………………..9

8. Cells pleomorphic becoming coccoid with age. Gram reaction ofbacilli and cocci usually positive....................................................………………Nocardia

Gram-positive coccoid cells in older cultures. Coccoid cellsgerminate to produce bacillus-shaped cells. Bacilli may beGram negative with Gram positive granules..............................……………..Arthrobacter

9. Catalase positive ........................................................................………………….CorynebacteriumCatalase negative .............................................................................…………………..Lactobacillus

10. Conidia or sporangia formed within one week ..................................................……………...11No conidia formed, anaerobic or microaerophilic ...........................………………Actinomyces

11. Chains of conidia formed; colony may produce brown watersoluble pigment and have an “earthy” smell ............................……………..Streptomyces

Single conidia only.............................……………………Micromonospora or Thermoactinomyces

12. Cells arranged singly, or in chains or clusters................................................…………………13Cells in cubical packets..............................................................................……………………….15

13. Catalase negative...........................................................................................……………………..14Catalase positive ............................................................................................……………………15

1 Endospores are seen within the vegetative cells on an endospore stain after growing on sporulation agar for 48hours.

2 Rod shaped cells break up into coccoid shapes or conidia after growing on an agar plate for several days

43

14. Large, mucoid colonies on sucrose agar; microaerophilic orfacultative anaerobic; capsule present.......................................………………..Leuconostoc

Small, round (1-2 mm) colonies on sucrose agar, no capsule.........………………Streptococcus

15. Glucose fermented .......................................................................…………………..StaphylococcusGlucose not fermented ..........….........................................................……………….. Micrococcus

16. bacillus shaped...............................................................................................…………………….17coccus shaped .......................................................................................…………………...Neisseriaovoid .................................................................................................……………………Azotobacter

17. Red or purple pigmented colonies on agar plate.............................................………………. 18No red or purple pigmented colonies; not associated with root

formations in plants.................................................................................…………………..19No red or purple pigmented colonies; associated with root

formations in plants. .................................................................................………………... 33

18. Acid from mannitol, pigment soluble in acetone:alcohol ................…………...…....... SerratiaNo acid from mannitol.............................................................………………….Chromobacterium

or Rhodopseudomonasor Rhodospirillum

19. Organisms produce a green, blue, brown or yellow water-soluble pigment which diffuses into the medium. Glucoserespired; oxidase positive; aerobic; motile............................………………...Pseudomonas

No water-soluble pigment produced.................................................................………………..20

20. Curved or bent bacilli on Gram stain...............................................................…………………21Straight bacilli................................................................................................……………………..22

21. Bent bacilli, methyl red (-), Voges Praskauer (+), catalase (+)......................……………..VibrioSpiral bacilli, no growth in peptone water (indole broth)

without cellulose strip....................................................................…………………Spirillum

22. Glucose not utilised...........................................................…………………................................ 23Glucose utilised facultatively.......................................................................………………….... 25Glucose utilised aerobically..............................................................................………………….28

23. Yellow pigmented colony.............................................................…………………FlavobacteriumNon-pigmented colony......................................................................................…………………24

24. Litmus milk alkaline, oxidase positive, aerobic, motile….…………….................... AlcaligenesLitmus milk alkaline, oxidase negative, non-motile…......................……………..Acinetobacter

25. Lactose fermentation produces acid.................................................................…………………26No acid from lactose........................................................................................…………………...29

26. Methyl red (+), no growth on citrate, fecal odor on BHI......................……………..EscherichiaMethyl red (-), growth on citrate......................................................................………………….27

27. Non-motile...........................................................................................…………………….KlebsiellaMotile..............................................................................................……………………..Enterobacter

28. Yellow pigmented colonies..........................................................……………….....XanothomonasNon-pigmented colonies....................................................................………………….Acetobacter

29. Urease (+)........................................................................................................…………………….30Urease (-).........................................................................................................…………………….31

44

30. Motile................................................................................……………………............................... 31Non-motile.............................................................................................…………………….Shigella

31. Indole (+), swarming growth on BHI agar..................................................………………ProteusIndole (-), no swarming growth.........................................................................………………...32

32. Limus milk acid.................................................................................……………………SalmonellaLitmus milk acid and peptonised........................................................………………...Aeromonas

33. Citrate positive.....................................................................................…………………..RhizobiumCitrate negative..............................................................................………………….Agrobacterium

Once your outline has been approved, carry out your tests using materials available in your kitsor on the side bench. You will have four lab periods.

Record all of your results in your lab book. Include diagrams of all staining results, as well asdescriptions of cell and colony morphology and tables of biochemical test results. Include the

results from Part C below.

Use reference material to identify your organism to species (note, identify all possiblespecies).

Note: you will be pooling your results with those from the other classes and examining class

results as well.

C Investigation of Catabolic Ability of Soil and of Compost Bacteria

This exercise will be completed concurrently with exercise B, above.

MethodsEach bench will require:

• 2 casein agar plates• 2 NA plates containing carboxymethylcellulose

• 2 NA plates containing starch• 2 NA plates containing xylan

• Each individual will require:• 1 casein agar plate

• 1 NA plate containing carboxymethylcellulose• 1 NA plate containing starch

• 1 NA plate containing xylan• 4 replica-plating templates per bench

• sterile toothpicks – 1 beaker per bench• waste beakers for used toothpicks

• Enumeration plates (of soil and of compost bacteria)

Each group of 4 is responsible for replica-plating 40 random colonies from plates of soil orcompost bacteria onto plates containing one of the 4 substrates of interest

45

(carboxymethylcellulose, starch, casein or xylan). Note that plates containing CMC, starch or

xylan all contain these substrates added to a nutrient agar base whereas plates containingcasein are composed of skim milk and agar only.

1) Using a sterile toothpick for each new colony, carefully scrape a well-defined colony

from one of your soil or compost bacteria enumeration plates.2) Stroke the toothpick across square number 1 on each of the three different labelled

plates (CMC, starch or xylan). For plates containing casein as the substrate, you mayneed to estimate placement of the colonies as you may not be able to see the template

through the plate. Place the used toothpick into the beaker provided.

3) Select a fresh toothpick and repeat steps 1-2 for 40 different colonies of bacteria.

Work individually to determine the catabolic ability of your unknown:

4) Obtain 4 plates, each containing a different substrate. Label with your name and

with the name of the substrate.5) Use an inoculating loop to streak out your unknown (from your PYE plate of pure

culture) onto each of the 4 plates (you want to streak for single colonies).

Invert all of the plates. These plates will be incubated at 30oC for 48 hours.

The Next Laboratory Period - Evaluation of Catabolic Ability:

6) For those plates containing xylan or carboxymethylcellulose as substrates, flood the

plate with a 0.1% (w/v) aqueous solution of Congo Red. Incubate for at least 5-10minutes, then pour off the excess solution into a waste beaker (not down the drain!),

and flood the plate with 1M NaCl to destain. Swirl the plate and let stand. Over thenext 30 minutes, perform this destaining step 2 more times. Be generous with the

NaCl. Cellulase- or xylanase-producing colonies will be surrounded by yellowhaloes visible against the red or orange background. If there are no obvious haloes,

then score the results as negative.7) For those plates containing starch as a substrate, flood the plate with a 0.13%

iodine/0.3% potassium iodine solution. Swirl to cover the surface of the plate, thendiscard immediately. Destain by flooding the plate with 1 M NaCl and allowing the

plates to stand. Amylase positive colonies should be surrounded by a zone ofclearing.

8) For those plates containing casein as a substrate, examine the plate closely.Caseolytic positive isolates should be surrounded by zones of clearing.

Thought Questions:

46

• For enumeration, why do you only count plates having between 30 and 300 colonies?• Why do you incubate soil and compost bacteria at 28-30oC?

• Provide one specific example of a differential medium used in the current exercise.• What is the differential component in the medium in your answer in (a)?

• How is the medium differential?• Is this medium type selective also? Why or why not? How would you make this

medium selective for the carbon source in question?• In addition to manipulating nutrients, how else could you make culture conditions

selective? Provide a specific example.

References:

Atlas, R.M. and Richard Bartha. 1998 Microbial Ecology: Fundamentals and

Applications, Fourth Edition. Benjamin/Cummings Publishing Company, Inc.640 pp.

Poulsen, O. M. and Petersen, L. W. 1989. Electrophoretic and enzymatic studies on the crude

extracellular enzyme system of the cellulolytic bacterium Cellulomonas sp. ATCC21399.Biotechnol. Bioeng. 34: 59-64.

Ross, H. 1993. Cellular, Molecular and Microbial Biology 343 Laboratory Manual. The

University of Calgary press, Calgary AB.

Teather, R. M. and Wood, P. J. 1982. Use of Congo red polysaccharide interactions in theenumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl.

Environ. Microbiol. 43: 777-780.

47

EXERCISE 9

APPLICATIONS OF MICROBIOLOGY

A number of industrial processes make use of the end products of bacterial and fungalfermentations. For instance, in the presence of acid producing bacteria, and often the enzyme

renninase, milk will form curds (solid) and whey (liquid). Once the solids are compressed,salted, and aged, the resulting product is cheese. Different cheeses are produced by varying

the bacterial inoculum, varying the milk used, or even by introducing fungi such as certainspecies of Penicillium into the curds.

Some Streptococcus species and some Lactobacillus species produce only lactic acid as a result

of reduction of pyruvic acid. These organisms are responsible for the production of yogurt.Yogurt can be made from milk simply by inoculating with a starter culture of yogurt that

contains live bacterial culture. Conversely, yeasts produce alcohol and CO2 rather than lacticacid as a result of the reduction of pyruvic acid.

EXPERIMENTAL OBJECTIVE

This experiment will illustrate fermentation pathways and organisms involved in the

production of alcohol.

A Alcohol Production

Prior to your lab period, grape juice, water and yeast cells were added to a sterile container. Over

the next two weeks, you will be responsible for sampling the fermenting juice at various time

intervals. The primary fermentor is inoculated with a high cell density (~106 yeast cells/mL).

The bulk of the must (grape juice medium) is rapidly depleted of oxygen by the yeast and

remains anaerobic, despite the primary fermentor remaining open to the atmosphere. Yeast cells

continue to reproduce by acquiring the needed energy and carbon through fermentation. The

fermentation is an ethanolic fermentation because ethanol and CO2 are the fermentation

endproducts.

Growth of the yeast culture can be monitored by measuring optical density and enumerating

CFU/mL (Recall Exercise 4 - Bacterial Reproduction). Ethanol concentration can be estimated

indirectly by measuring the specific gravity of the wine must with a hydrometer. The specific

gravity of the must decreases as the grape juice sugars are converted to ethanol and CO2. A

"specific gravity to percent ethanol" conversion chart supplied with the hydrometer is then used

to determine ethanol content of the must.

Note: this exercise requires some out of lab participation. Failure to sign up will result in a

deduction of 5% from your lab grade.

48

Please sign up for a time slot when at least half of your group members can attend.

MATERIALS (in C741)

• Wine thief

• Spectrophotometer (warm up 15 minutes prior to reading OD values)

• Primary fermentor

• pH paper

• Micropipettors and sterile tips

• Sterile microfuge tubes

• Rack for microfuge tubes

• Filter sterile wine must for diluting samples

• Bunsen burner

• Hydrometer

• Sterilising solution

• Graduated cylinder

• Thermometer

• 28 oC incubator

• YPD plates

• Spreaders and alcohol

• Spreadsheet for recording results

At each time point the following data must be collected by each group:

• Temperature

• OD600

• pH

• Specific gravity

• Viable counts

Procedure – Work very carefully. These results will form the basis of your major lab report.

1) Mix the culture well, then remove a sample from the primary fermentor.

2) From the sample (not the primary fermentor), measure and record the sample

temperature.

3) Measure and record the specific gravity.

4) Measure and record the pH of the sample.

5) Measure and record OD600. Use the blank provided to zero the machine. Read the optical

density of at least 5 mL of the sample. If the OD600 exceeds 0.7, you will have to dilute the

sample with the sterile must provided (start by creating a 1:1 dilution). Read the OD600 of

the diluted sample, then multiply by the dilution factor to obtain your corrected reading.

Record the corrected reading on the sheet provided.

49

Please save the blank for the next group.

5) For viable counts, Table 8.1 provides you with dilutions to create depending upon your

sample time, as well as guidelines for what dilutions to plate out. Ensure that you plate

out duplicates of each dilution.

6) For your dilutions, prepare the required number of 900 µL dilution blanks = 900µL of

filter sterile wine must in 1.5 mL microfuge tubes.

7) Clearly label plates with time, name and dilutions. Spread plate (in duplicate) 100 µL of

suggested dilutions on YPD agar.

8) Invert plates and incubate at 28ºC for 48 hours.

9) Tidy up work area.

Thought Questions:

• Numerous data relating to alcohol fermentation were collected by the class over the sampling

period, including measurements of pH, temperature, specific gravity, optical density, and

CFU’s/mL of culture. Design and construct a series of figures to graphically represent the

data that were collected.

• Calculate the generation time and the specific growth rate of the yeast cells in the culture.

• Name two factors that control the final ethanol concentration in a culture.

• Although we stirred our culture each time before sampling, winemakers do not. Why would

winemakers not stir the culture?

• Why did the pH of the culture change as fermentation proceeded?

• Why did the specific gravity of the culture change over time?

50

Table 8.1 Dilutions of yeast mixture to create and to plate out for all time points.

Time point

(hours)

Dilutions to Create Dilutions to Plate

2 None

Spread plate 100 µL of filter

sterile wine must in duplicate on

YPD

3 10-3; 10-4; 10-5

Spread plate 100 µL of each of

the 3 dilutions in duplicate on

YPD

7 10-3; 10-4; 10-5

Spread plate 100 µL of each of

the 3 dilutions in duplicate on

YPD

12 10-3; 10-4; 10-5

Spread plate 100 µL of each of

the 3 dilutions in duplicate on

YPD

24 10-4; 10-5; 10-6

Spread plate 100 µL of each of

the 3 dilutions in duplicate on

YPD

28 10-4; 10-5; 10-6

Spread plate 100 µL of each of

the 3 dilutions in duplicate on

YPD

32 10-4; 10-5; 10-6

Spread plate 100 µL of each of

the 3 dilutions in duplicate on

YPD

48 10-4; 10-5; 10-6

Spread plate 100 µL of each of

the 3 dilutions in duplicate on

YPD

53 10-4; 10-5; 10-6

Spread plate 100 µL of each of

the 3 dilutions in duplicate on

YPD

79 10-4; 10-5; 10-6

Spread plate 100 µL of each of

the 3 dilutions in duplicate on

YPD

217 10-4; 10-5; 10-6

Spread plate 100 µL of each of

the 3 dilutions in duplicate on

YPD

51

APPENDIX 1THE COMPOUND LIGHT MICROSCOPE

As you label Figure 1, your Instructor will review the use of this microscope with you. Locate the

ocular lens (eyepiece); there will be one if the microscope is monocular, or two if it is binocular.Then locate the objective lenses, the ones nearest the object to be studied. These two lenses

(ocular and objective) are connected by the body tube of the microscope. The objective lenses(there will be two or more, the smallest being that with the least magnifying power, and the

largest being that with the greatest magnifying power) are mounted on a revolving nosepiece

above a flat stage on which the study specimen (slide) is placed.

Figure 1: The Compound Microscope

Your microscope is equipped with a mechanical stage. This consists of a clip to hold the slide in

place (the clip is spring-loaded; the Instructor will demonstrate how it works) and two knobs atthe side of the microscope body to move the slide side-to-side, or forward-to-back. Note also the

two micrometer scales on the mechanical stage, which allow you to note the coordinates of aparticular object on the slide you are viewing.

52

Place a slide on the stage and center it over the hole in the stage. Adjust the distance between the

oculars to match your interpupillary distance (distance between your pupils). Revolve thenosepiece so that the lowest power objective lens (generally the 10x power lens) is in position. To

focus the microscope, locate the coarse and fine adjustment knobs at the base of the microscope,and use the coarse adjustment to move the slide close to, but not touching, the objective lens.

Look at the stage from the side as you do this. On most microscopes this involves raising thestage, but on some the lenses are lowered. Also, on most microscopes an automatic stop will

prevent you from moving the stage closer than about one centimeter from the lens. Now, lookthrough the ocular lenses, and move the slide away from the objective lens until the specimen

becomes clear (is in focus). Finish focusing with the fine adjustment knob. Once you havefocused with the low objective power lens, you may switch over to the next higher power lens

with only fine focus adjustments (the microscope is said to be parfocal).

As you switch from one objective lens to another, you will notice that the working distance, theclearance between lens and stage, decreases with increasing lens power. This is illustrated in

Figure 2 below.

Figure 2: The working distance (above) and the field of view (below) changewith magnification of objective lens.

It should be obvious to you why, on high power objective lenses (40x or 100x), you must use only

the fine focus knob to adjust focus; otherwise the risk of (damaging) contact between lens andslide becomes great. Also illustrated in Figure 2 is the diminishing field of view as objective lens

power increases; this is due to a smaller and smaller aperture at the bottom of the lens throughwhich light enters. This means that [a] things are harder to find on a slide when you are using

high power since only a small fraction of the slide can be seen, and [b] less light enters your eyeand everything in the field appears darker. As a consequence, you will learn to [a] switch back to

53

a lower power objective lens when you want to "scan" around the slide, and [b] manipulate the

amount of light coming into the lens so that you can see the objects clearly.

The amount and concentration of light coming through the specimen and hence to your eye canbe adjusted in several ways. First, of course, is the on/off light switch, generally located at the

base of the microscope, and often associated with a rheostat to control light intensity. Acondenser lens is mounted below the stage, and concentrates the light on to the specimen; it

generally needs no adjustment of position. An iris diaphragm is located below the condenserlens. Find the lever which controls the diaphragm; it can be very useful in adjusting illumination

and contrast.

Biology 3200 microscopes are binocular, containing two eyepieces. To correct for the slightdifference in the focus of your two eyes, precisely fine focus a specimen using only your one eye

which is at the non-focusing ocular (if your microscope contains two focusing oculars, either maybe used to begin). Next, open the other eye and bring the image into focus for that eye using only

the ocular focus. Since other students use these same microscopes during the semester, thisexercise of binocular focusing should be performed at the onset of each microscope session.

Finally, some useful hints and cautions:

• Never drag the microscope across the counter-top. Lift it with both hands by its arm, beingcareful not to tip it.

• Use lens paper to clean glass slides and lens surfaces before using your microscope.• Water damages objective lenses; if water does contact a lens, wipe it off immediately. Also

avoid getting water under the slide as it will stick to the stage.• If you have used immersion oil, use lens paper dipped in 60 % ethanol to remove it from the

100x objective lens when you are finished.• Always start the focusing procedure with low (10x) power lens.

• When attempting to locate an object on a slide, remember that the image you see is reversed;that is, as you move the slide toward you on the stage, the slide is apparently moving away

from you as you view it through the lens.• Some ocular lenses are equipped with pointers; they appear as a dark black line that will

rotate if the lens is rotated in its tube.

Electron Microscopy

Bacterial size places them at the limits of resolution of the light microscope. Even with the bestquality lenses, magnification can only be increased slightly beyond 1,500x. Much higher

magnification can be achieved with the electron microscope with the scanning electron

microscope (SEM) reaching about 100,000x magnification and the transmission electron

microscope (TEM) capable of 1,000,000x magnification.

54

APPENDIX 2PREPARATION OF SCIENTIFIC DRAWINGS

1) Use a sharpened pencil; never ink. The lead should be hard.2) Place drawing to one side, usually the left, leaving room for labels to the right.

3) Try to draw with one continuous line and do not retrace your lines. Do not shade.4) Place label lines horizontally (use a ruler), with no crossed lines.

5) Objects labelled should be singular unless label line branches to multiple objects.6) Label only what you see, not what you think should be seen.

7) Below the figure you should add:a) The title of the diagram

b) The magnification of the drawing (see below)8) The magnification of the diagram gives you the relationship between the size of your

diagram and the actual size of the specimen. A diagram of a cell would be much largerthan the actual cell, whereas a diagram of an elephant could be much smaller than the

actual elephant.

Magnification is defined as: size of drawingactual size of specimen

Where:• size of the drawing is measured with a ruler

• actual size of specimen is determined by one of the methods in Exercise 1.• the number calculated has as many significant figures as the accuracy of your measurement

(usually 2, if you measure in mm)

55

9) Example of a drawing:

Figure 1. A chain of Bacillus subtilis cells stained with methylene blue (23 000x)

• Notice that in the figure, enough organisms are shown such that the arrangement can beseen.

• Drawing magnification is calculated based on length or width, not both of only one of theorganisms (not the whole chain).

• Figures are given numbers - Figure 1, Figure 2, etc.• As much detail as possible is provided in the title (eg Gram reaction seen, type of stain used,

type of organism etc.).

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APPENDIX 3ASEPTIC TECHNIQUE

A. Aseptic Technique

Much microbiological work, and to some extent biochemical work, depends on the maintenance

of pure cultures of microorganisms. Therefore, there are various essential precautions that MUSTbe observed to exclude unwanted organisms. Accidental contamination may ruin your results

completely.

Aseptic technique is largely a matter of common sense, but it is essential to realise that bacterialand fungal spores are present everywhere, and a high standard of technique must be attained.

Correct methods of handling cultures and apparatus will be demonstrated. These methods

should be followed.

Consider carefully and remember the following points:

1. Clean air contains many bacterial and fungal spores carried on dust particles or in waterdroplets. Any surface exposed to air quickly becomes contaminated, and if material is to

be kept sterile it should be exposed only as much as is absolutely necessary formanipulation. Instruments which can be sterilised by heating in a bunsen flame (e.g.

inoculating loops) can be left exposed, but they must be flamed thoroughly before use,and again before being replaced in the holder.

Items of equipment that cannot be treated in this way (e.g. pipettes) are sterilised in

wrappings or containers from which they must not be removed until actually needed.They must not be allowed to touch unsterile surfaces during use. Plugs and caps of

tubes and bottles must not be laid on the bench nor must sterile containers be left

open to collect falling dust.

2. Clothes, hair, skin and breath all carry a heavy microbial load and where strict asepsis is

essential, sterilised gowns, caps, gloves etc. are worn. Even in normal microbiologicalwork care must be taken to prevent contamination from the above mentioned sources. A

clean laboratory overall is advised for all lab work.

Microbial contamination in the lab is most often due to currents of unsterile air. The chief meritof inoculation chambers and screens therefore lies in the protection they give from drafts. This

protection can be supplemented by keeping all windows and doors shut and by cutting downpersonal movement within the laboratory. These precautions can be offset by careless use of

burners that create convection currents.

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3. Before any operation is started, all necessary materials should be assembled in

convenient order with provision for protecting sterile objects until needed, and fordisposing of used apparatus (so as not to contaminate other material).

B. Aseptic Culture Manipulation

Purposes: 1) To prevent the contamination of the environment and people working in the

laboratory from the cultures used in the exercises2) To prevent accidental contamination of cultures of microorganisms and of

solutions and equipment used in the laboratory

Correct methods of handling cultures and apparatus will be demonstrated. These methods

should be followed. Consider carefully and remember the following points:

• Prior to starting any work in the laboratory, wash hands with soap, and wash down

bench area using 10% bleach. This procedure should be repeated after the lab iscomplete.

• Avoid working on your lab book or lab notes.

• Clean laboratory coats must be worn. If you have long hair, tie it back before working in

the laboratory environment.

• Eating or drinking are not permitted in the laboratory. Do not place pencils, fingers oranything else in your mouth.

• Clean air contains many bacteria and fungal spores carried on dust particles or in water

droplets. Any surface exposed to air quickly becomes contaminated. If material is to bekept sterile, it should be exposed only as much as is absolutely necessary for

manipulation.

Plugs and caps of tubes, tops of Petri dishes and bottles of solutions, (even water!!) must not

be laid on the bench nor must sterile containers and cultures be left open and exposed to the

air.

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Inoculation of Culture Tubes

Again, the important thing to remember is that exposure of sterile liquids or bacterial cultures to

air must be minimised.

-Ensure that you have the tubes, plate of inoculum, inoculating loop and a sterile tube of mediumavailable within easy reach.

-Flame the inoculating loop until red hot. When removing inoculum from a tube, remove the capfrom the tube by grasping the cap between the last finger and the hand which is also holding the

inoculating needle (Figure 1). Do not place the cap on the bench!!

Figure 1: Technique for manipulating test tubes aseptically.

-Flame the mouth of the tube by passing it rapidly through the Bunsen burner 2-3 times. This

sterilises the air in and immediately around the mouth of the tube.-Cool the loop on the inside of the tube, remove the inoculum.

-Reflame the mouth of the tube and replace the cap-Flame the inoculating loop before replacing

-Note, when removing inoculum from a plate, cool the loop in the agar before picking up thebacteria

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Streaking for Single Colonies

-A loop of liquid culture or a small amount of bacterial growth from a plate culture is transferred

aseptically to a sterile plate in the area shown by Figure 2A.-Once the first set of streaks has been made, the inoculating loop is reflamed until red hot. DO

NOT REINTRODUCE THE LOOP INTO THE ORIGINAL CULTURE!!!

-Cool the loop, and make a second set of streaks as shown in Figure 2B, only crossing over the

initial set of streaks once.-Flame the loop again, cool, and repeat for three more sets (Figure 2C). Note, try not to gouge the

agar while streaking the plate.

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Preparation of Spread Plates:

Generally, volumes of culture greater than 100 µµµµL are NOT plated as it takes too long for the

liquid to dry.

• Use aseptic technique to obtain 100 µµµµL of culture and place in the middle of a plate of

medium.

• Use a sterile glass spreader (this may involve dipping a spreader into a beaker of alcohol

and waving it through a Bunsen Burner flame. If this is the case, DO NOT hold the

spreader in the flame and avoid tipping the spreader so that flaming alcohol runs over

your hand. Once the flame has burnt out, the spreader is ready to use).

• Use the same hand that holds the spreader to lift the lid of the plate and keep it just above

the plate the entire time.

• Gently touch the spreader to the side of the medium (not directly in the culture in case the

spreader is still a bit warm). Smooth the culture evenly over the surface of the plate

ensuring that you cover the entire plate.

• Invert the plates and place in the incubator when dry.

C. Sterilisation

Media must be sterilised after distribution into tubes, flasks or bottles. Sterilised media may laterbe transferred aseptically to previously sterilised containers, but this should only be done when

really necessary, e.g. in preparing "plate" cultures, since some risk of contamination isunavoidable.

Methods of Sterilisation

1. Most media (including agar) can be sterilised by treatment with steam under pressure in

an autoclave, the usual treatment being 15-20 minutes at a pressure of two atmospheres.This raises the steam temperature to 121°C. When using an autoclave, the water should

be allowed to boil, and the steam to fill the autoclave before shutting the valve. Thisallows the material to heat up and ensures that the correct steam pressure is attained.

Never overfill an autoclave since this will upset the pressure/volume relationship andthe correct temperature will not be attained. Materials that might be adversely affected

by this treatment may sometimes be treated for a short time or at a lower temperature,but this will not be effective if the material is heavily contaminated to begin with. Screw

caps on bottles must be left slightly open during sterilisation and screwed down onremoval from the steriliser.

2. Media that are difficult or impossible to autoclave satisfactorily, e.g. gelatin media and

some sugar media, may be sterilised by intermittent steaming. Objects to be sterilised areheated over boiling water in a steamer (steam temperature 85°-95°C) for 15-20 minutes

on each of three or more successive days. Time must be allowed for the medium to reachthe same temperature as the steam. Between treatments the material must be kept at a

temperature allowing spores to germinate (30°-37°C) and so lose their heat resistance.

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3. It is often necessary to sterilise some ingredients of a medium separately and to add themto the rest of the medium before use. Heat-labile ingredients, e.g. urea, serum, etc. must

be sterilised by filtration through a bacteria-proof filter, i.e. Seitz filters or membranefilters.

4. Dry glassware, e.g. glass petri dishes, empty flasks, pipettes may be sterilised in theautoclave and then dried or may be sterilised in a hot air oven. Any oil material

must also be sterilised in a hot air oven. The minimum effective treatment is 1 hourat 150°C. This should be increased to 160°C or the time of heating prolonged to 2 or 3

hours wherever possible.

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APPENDIX 4

THE CULTIVATION OF BACTERIA

(Please read Madigan et. al., 2003; Chapter 5)

In order to grow, microorganisms require a) water, b) macronutrients eg. – C, N, K, P, S, Mg, Ca,

Na, and Fe c) micronutrients (trace elements) eg. - Fe, W, Zn and d) growth factors – vitamins,amino acids, purines and pyrimidines.

In general, wild-type organisms are termed prototrophs. An auxotroph is a nutritional mutant,

unable to synthesise an essential component for growth from precursors. Note that this essentialcomponent is normally synthesised by the wild-type or prototrophic strains of the same species.

Scientists study and manipulate nutritional requirements of bacteria or yeast using minimal

media. Minimal or defined media are those in which the exact chemical composition of all

ingredients is known. A medium where the exact chemical composition is not known is termedcomplex. Complex media are preferred as they are generally easier to prepare than minimal

media, they result in high levels of growth, and are useful when exact nutritional requirements ofan organism are not known.

Nutritional Classification:

The nutritional classification of organisms is based on three parameters: the energy source, the

principal carbon source and the source of reducing power. With respect to energy source,phototrophs are photosynthetic organisms that use light as their energy source and chemotrophs

are organisms that depend on a chemical energy source. Organisms able to use CO2 as aprincipal carbon source are autotrophs. Heterotrophs depend on an organic carbon source. To

designate the source of reducing power, the term lithotroph or organotroph is applied.Lithotrophs use inorganic compounds as their source of reducing power, and organotrophs use

organic compounds as their source of reducing power.To summarise:

source of energy source carbon source reducing power

photoautotroph light CO2 inorganic(photolithotroph) oxidizable

substratephotoheterotroph light organic organic

(photoorganotroph)chemoautotroph chemical (oxidation of CO2 inorganic

(chemolithotroph)* reduced inorganiccompounds e.g. NH3,

NO2- and H2)chemoheterotroph chemical organic organic

(chemoorganotroph)

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*All chemoautotrophs are chemolithotrophs, but not all lithotrophs are autotrophic. For

example, the methylotrophic bacteria can use organic carbon as their carbon source.

Common Media Constituents (see Table 5.4, Madigan et. al (2003) for examples):

Energy or Carbon sources:

• Sugars, alcohols, carbohydrates and amino acids

• Found in infusions – for instance – beef infusion• Found in extracts – for instance – yeast extracts

• Also found in peptones (see below)

Nitrogen sources:

• Inorganic sources such as ammonia or nitrate

• Nitrogen fixing organisms use atmospheric N2

• Extracts, infusions

• Peptone – hydrolysis of proteins produces mixtures of short-chains of amino acids(peptides). Sources of peptones may include meat, fish, blood, or soybeans

• Tryptone – pancreatic digestion of casein

Other Macronutrient Source Examples:

• MgSO4

• CaCl2

• Potassium salts

Micronutrient Sources:

• May not be necessary to add as these are required in such small concentrations.

Growth Factors:

• Some organisms are able to synthesise all growth factors from precursors. Other

organisms require these compounds already synthesised• For example – thiamine, biotin

Buffering Components

Buffers, which prevent large changes in pH, are often required to facilitate growth. This is

particularly true of media composed of simple compounds or in which acid-producing bacteria

are cultivated. Mixtures of sodium and potassium phosphates are often employed. In complex

media, buffering is provided by the peptides and amino acids.

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Gelling Agents

For a solid medium, agar, a water soluble polysaccharide, is added to the medium. First

discovered in 1658 in Japan, agar was first used for microbiological purposes by R. Koch in 1882.It is extracted from members of Class Rhodophyceae (a group of red-purple marine algae). Agar is

particularly suited to microbial propagation because:• It lacks metabolically useful chemicals such as peptides and fermentable carbohydrates

(it cannot be broken down by bacterial enzymes)• It melts at a high enough temperature (85 oC) to support growth of different temperature

requiring microbes• It lacks bacterial inhibitors

Below are two examples of media used for cultivation of microbes. TY is an example of a

complex medium whereas VMM is an example of a minimal or defined medium:

TY Agar (used for the cultivation of organisms such as Rhizobium leguminosarum,

Pseudomonas fluorescens)

As with most complex media, ingredients for TY are weighed out, 1 L of water is added,and the mixture autoclaved. After cooling slightly to approximately 60 oC, TY medium is

poured into Petri dishes.

Ingredient Amount (/L) Source of?

Tryptone 5.0 g Macronutrients (primarily

nitrogen, also carbon andgrowth factors in the form of

amino acids)

Yeast Extract 3.0 g Macronutrients (primarily

carbon, also nitrogen andgrowth factors)

CaCl2 0.5 g Macronutrients

MgSO4 0.1 g Macronutrients

Agar 20 g Gelling agent

For the next example – VMM – three different mixtures (Solutions A, B and C) of ingredients

are made up separately, autoclaved separately, and then combined. Finally, a carbon source is

added just prior to pouring.

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VMM (Vincent’s Minimal Medium - Vincent, 1970) (used for the study of nutritional

requirements of Rhizobium leguminosarum)

Solution A:

Compound Amount (/L) Source of?

K2HPO4 1.0 g Buffering agent/Macronutrients

KH2PO4 1.0 g Bufferingagent/Macronutrients

KNO3 0.6 g Macronutrients (nitrogen inparticular)

For Solid Medium:Agar

12.5 g Gelling agent

Solution B (10x):

Compound Amount (/L) Source of?

FeCl3 0.1 g Macro/Micronutrients

MgSO4 2.5 g Macronutrients

CaCl2 1.0 g Macronutrients

Autoclave and add to a final concentration of 1x

Solution C (100x)

Compound Amount for: 1 L Source of?

Biotin 0.01 g Growth factors

Thiamine 0.01 g Growth factors

Calcium Pantothenate 0.01 g Growth factors

Autoclave and add to a final concentration of 1x.

Carbon sources: Depending on the organism studied, a variety of carbon sources may be added.For instance, when studying genes required for catabolism of a certain carbon source, a scientist

will often first create a mutant or auxotroph unable to catabolise that carbon source. To confirmpresence of the mutation, it is necessary to plate the putative auxotroph on medium containing

the carbon source of interest, and plating on a medium containing a carbon source that theorganism is able to utilise. In Rhizobium leguminosarum, some examples of carbon sources that are

useful for these types of experiments are mannitol, sorbitol (both are sugar alcohols), orrhamnose. Each carbon source is prepared as a stock solution, filter sterilised, and added to a

final concentration of 0.4% (w/v).

Oxygen Requirements of Microorganisms.

Many species of bacteria are facultative aerobes, i.e. they can grow under aerobic or anaerobicconditions, the latter ability being dependent upon the presence of some substance that can be

utilised as an electron acceptor by the species concerned. Some bacteria are obligate aerobes,unable to use anything but oxygen as a final electron acceptor. Others are obligate anaerobes that

cannot use oxygen as an electron acceptor. A few bacteria are somewhat intermediate, growing

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best in low oxygen tensions. These are called microaerophilic bacteria. During growth in liquid

culture, microorganisms tend to utilise all available oxygen and so reduce the medium. Thus, theoxidation-reduction potential (Eo) of the medium may become low enough to allow anaerobic

growth to occur. One example of this is found in the fermentation of sugar to produce alcohol byyeast (Exercise 8 part C). Unless the mixture is stirred frequently, the little oxygen available in

the grape juice solution is utilised rapidly by the growing culture. Organisms then switch toanaerobic growth.

In order to sample material containing anaerobes, specimens must be obtained and immediately

placed into an environment containing an oxygen-free gas and an indicator that changes colourwhen oxidised to indicate when oxygen has contaminated the sample. Organisms may then be

cultured in sealed jars containing gas mixtures of N2 and CO2 or even by cultivation in ananaerobic chamber.

Temperature Requirements of Microorganisms

Cultures should be incubated at the temperature most favourable to growth or the specific

activity being studied. Human pathogens and commensal species grow best at bodytemperature, i.e. 37°C. Soil organisms and plant pathogens are normally incubated at 20-30°C.

The optimum temperature is that temperature at which the growth rate is maximal for aparticular organism. Note that for every organism, there is also a minimum temperature below

which no growth occurs, and a maximum temperature, above which no growth occurs.

The terms used to describe microorganisms according to their temperature requirements are asfollows:

• thermophiles require temperatures of 45°C-65°C

• extreme thermophiles (which are usually archaebacteria) will grow at temperatures above65°C.

• mesophiles grow best at temperatures of 20°C-45°C.

• psychrophiles require low temperatures - below 15°C.

References:

Difco Manual. 1998. Difco Laboratories, Division of Becton Dickinson and Company,Maryland.

Madigan, M. T., Martinko, J. M., and Parker, J. 2003. Brock Biology of Microorganisms 10th

Edition. Prentice-Hall Canada Inc., Toronto.

Ross, H. 1992/3. Microbiology 241 Lab Manual. University of Calgary Press, Calgary.

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APPENDIX 5BACTERIAL OBSERVATION

Bacterial genera may be differentiated in two ways:1) by the cellular morphology which is observed microscopically

2) by the colony morphology which is observed on a plate culture

Cellular Morphology includes:1) Shape: rods, cocci, spirilli2) Size (in µm): diameter (cocci); lengthxwidth (rods)

3) Typical arrangement of the cells: chains, clusters, pairs, random

4) Gram reactionA diagram drawn to scale accompanies the cellular morphology.

Colony Morphology is that of a single isolated colony on the plate, not the morphology of the

entire bacterial growth on the plate. Colony morphology is influenced by medium composition;type of medium organism is grown on (defined, complex, specific type) should be noted in

conjunction with the description of colony morphology.The following characteristics are those most commonly used to describe colony morphology:

1) Shape or form

Circular Irregular Rhizoid Filamentous Punctiform (1mm or lessin diameter)

2) Surface: smooth/rough; mucoid/moist/dry/powdery

3) Elevation:

Flat Raised Convex Umbonate Umbilicate

4) Size: measure a single colony with a ruler5) Pigment: cream, white or beige coloured organisms are usually considered to be non-

pigmented. Pigments may be purple, red, pink, yellow, brown, blue, grey, etc. Watersoluble pigments diffuse into the medium.

6) Opacity: Transparent (can see through) or opaque.

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APPENDIX 6LABORATORY REPORTS

Lab reports shall be in the style of scientific papers published in refereed journals. This scientificstyle is relatively similar across journals although specific formats vary, including the form ofliterature citations. The journals Microbiology or Canadian Journal of Microbiology will be used asmodels for the specific format of Biology 3200 reports. Please do not use formats from journalssuch as Nature or Proceedings of the National Academy of Sciences as this will result in loss of marks.For detailed information on preparation of scientific reports, please refer to the Biology 3200 website.

The text should be in prose form and standard rules of grammar apply. Check spelling,including technical terms and names of bacterial species which are italicised or underlined; forinstance, Escherichia coli or Escherichia coli.

The reports shall be double-spaced, single-sided and typed. Staple the report together and do notsubmit it in a cover.

The reports shall contain the normal components of a scientific paper including:

Title - the title should identify the experimental topic as completely as possible.

Abstract - the abstract is an abbreviated version of the complete report. Typically containing nomore than 250 words, the abstract picks out the highlights of the introduction, methods, resultsand discussion. The abstract should be complete enough that it can be removed from the reportand will still provide a meaningful description of the study.

Introduction - The introduction serves to (i) provide background information and a descriptionof what is known prior to the study, and (ii) offer a justification for the study. This justificationdescribes why the experiment was performed - how does it fit into science and are there anyapplied aspects of the knowledge (i.e. is it relevant to medicine, agriculture or other disciplines).Relevant literature is used and cited.

Methods - The methods or 'Materials and Methods' describes the materials involved in the study,including biological materials (bacteria, etc.), and outlines the procedures used in the study.Reference must be made to this laboratory manual (Pacarynuk and Danyk, 2004). Otherreferences, the text by Madigan et. al., (2003), or other published materials may be cited. Globalreferencing (“All of the following methods are taken from…”) should be avoided. The methodssection should be adequate for the reader to completely understand what was done and also tobe able to repeat fully the study.

Results - The results describe the observations or experimental outcomes, providing figures,tables or other data as suitable. This section answers the question “What Happened?” The authorshould decide what is the most suitable format for experimental information and draft the reportaccordingly. Figures may include drawings that should be in pencil. Graphs or other figuresmay also be included as appropriate. Experimental results should be presented only once. If

69

information is presented in a figure then it should not be repeated in a table. Each figure andtable must have a caption which is complete enough that the figure and caption can be removedfrom the report and still be understandable. Figures and tables must be referred to in the textand described so that if the reader did not have the figure or table, trends or highlights of theresults would still be evident. Never include a figure or table without referring to it anddescribing it; to do so will result in loss of marks. Avoid evaluating or interpreting your resultsin this section.

Discussion - The discussion should refer to concepts or questions posed in the introduction andrelate these concepts from the literature to the results. Do not restate the results in this section.Your discussion will be graded based on your evaluation of the results with respect to theliterature. Any time you use information from another source, it must be immediately citedwithin the text. Failure to do this constitutes plagiarism and may result in a mark of zero beingassigned for the entire document. For examples of how to cite properly, refer to peer-reviewedjournal articles in Microbiology or Canadian Journal of Microbiology.

Never include quotations, such as phrases from the course text or this lab manual. Direct quotesare inappropriate in scientific writing. Always introduce relevant concepts using your ownwording and then cite using the format found in Microbiology or in Canadian Journal ofMicrobiology.

Literature Cited – This section only includes references cited within the body of the text.

Again, use the format found in Microbiology or the Canadian Journal of Microbiology.References will include journal papers, books and most likely, Holt (1989) or Holt (1994)

(Bergey’s Manual of Systematic Bacteriology). It is important to note that Bergey did not

write Bergey’s Manual of Systematic Bacteriology; the proper formats for referencing are as

follows:

Holt, J. G. (editor-in-chief). Bergey’s Manual of Systematic Bacteriology, Vol. I, 1984; vol. II,1986; vols, III and IV, 1989. Williams and Wilkins, Baltimore.

Holt, J. G. (editor-in-chief) (1994). Bergey’s Manual of Determinative Bacteriology, 9th edition.

Williams and Wilkins, Baltimore.

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APPENDIX 7USE OF THE SPECTROPHOTOMETER

Many procedures for the quantitative analysis of compounds in biological fluids are based on the

fact that such compounds will selectively absorb specific wavelengths of light. For example, asolution that appears red to us (such as blood) absorbs the blue or green colours of light, while

the red is reflected to our eyes. The eye, however, is a poor quantitative instrument, and whatappears bright red-orange to one person may appear dull red-purple to another. A

spectrophotometer is one instrument that will objectively quantify the amount and kinds of lightthat are absorbed by molecules in solution. A source of white light is focused on a prism to

separate it into its individual bands of radiant energy (Figure 1). One particular wavelength isselected to pass through a narrow slit and then through the sample being measured. The sample,

usually dissolved in a solvent, is contained in an optically selected tube or cuvette, which isstandardized for wall thickness and has a light path exactly one centimeter across (these tubes are

therefore expensive!).

Figure 1. A photoelectric spectrophotometer.

After passing through the sample, the selected wavelength of light strikes a photoelectric tube. If

the substance in the cuvette has absorbed any of the light, the light transmitted out the far side

will then be reduced in total energy content. When it hits the photoelectric tube, it generates an

electric current proportional to the intensity of the light energy striking it. By connecting the

photoelectric tube to a device that measures electric current (a galvanometer), a means of directly

measuring the intensity of the light is achieved. The galvanometer has two scales: one indicates

the % transmittance, and the other, a logarithmic scale with unequal divisions graduated from 0.0

to 2.0, indicates the absorbance.

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Zeroing the Spectrophotometer

Because most biological molecules are dissolved in a solvent before measurement, a source of

error can be the absorption of light by the solvent. To assure that the spectrophotometricmeasurement will reflect only the light absorption of the molecules being studied, a mechanism

of "subtracting" the absorbance of the solvent is necessary:1) Align the needle to 0 on the transmittance scale using the knob on the left hand side of

the machine (as you face the machine). Note, this step should be performed prior toplacing any tubes into the machine.

2) Insert the reagent "blank" (the solvent) into the instrument, and align the needle to 0 onthe absorbance scale using the knob on the right hand side of the machine (as you face

the machine). 3) The sample, containing solute plus solvent, is then inserted. Any reading on the scale

that is less than 100% transmittance (or greater than 0.0 absorbance) is considered to bedue to absorbance by the solute only.

Units of measurement: The transmittance scale is a % number; a ratio of the light exitingthe sample tube to the light entering the tube. However, this number is not a linear reflection of

the concentration of the solute molecules (Figure 2). The absorbance scale, on the other hand,does reflect a linear relationship. Although you do not necessarily know the exact concentration

of the solute molecules in your sample, you do know that if the absorbance value doubles, theconcentration of solute in your sample has doubled. Absorbance has no units, but the

wavelength of the light is usually indicated by a subscript.

Figure 2. The relationship between % transmittance and solute concentration (on the left), and

absorbance and solute concentration (on the right).

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APPENDIX 8Media, Reagents and pH Indicators

MEDIA:

Tryptic Soy Broth:

A general purpose medium used to cultivate a variety of microorganisms.

Composition (g/L):

Bacto tryptone 17.0 g

Bacto soytone 3.0 gDextrose 2.5 g

NaCl 5.0 gDipotassium phosphate 2.5 g

Dissolve in distilled water to a final volume of 1 L, dispense into test tubes, and autoclave

for 15 min at 121oC.

Tryptic Soy Agar:

Used for cultivation of a variety of microorganisms.

Composition (g/L):

Bacto tryptone 15.0 gBacto soytone 5.0 g

NaCl 5.0 gAgar 15.0 g

Dissolve in distilled water to a final volume of 1 L, autoclave for 15 min at 121oC, and

pour into sterile Petri dishes.

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LB Medium (Luria-Bertani Medium):

Used for cultivation of Enterobactereaceae family members, Sinorhizobium andAgrobacterium

Composition (g/L):

Tryptone 10.0 g

Yeast extract 5.0gNaCl 10.0 g

Dissolve in distilled deionised H2O to a final volume of 1 L, autoclave for 20 minutes at15 psi (1.05 kg/cm2) on liquid cycle, and pour into sterile Petri dishes.

Terrific Broth (TB)

Used for the cultivation of E. coli

Composition (g/L)

Tryptone 12.0 gYeast Extract 24.0 g

Glycerol 4.0 mL

Dissolve in distilled deionised H2O to a final volume of 900 mL, autoclave for 20 minutesat 15 psi (1.05 kg/cm2) on liquid cycle. Allow the solution to cool to 60 oC or less, and

then add 100 mL of a sterile solution of 0.17M KH2PO4, 0.72M K2HPO4 (this is thesolution resulting from dissolving 2.31 g of KH2PO4 and 12.54g of K2HPO4 in 90 mL of

deionised H2O. After the salts have dissolved, adjust the volume of the solution to 100mL with deionised H2O and sterilise by autoclaving for 20 minutes at 15 psi on liquid

cycle).

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Nutrient Agar:

Used for the cultivation of a wide variety of microorganisms.

Composition (g/L)

Peptone 5.0 gNaCl 5.0 g

Yeast extract 2.0 gBeef extract 1.0 g

Agar 15.0 g

Dissolve in distilled water to a final volume of 1 L, autoclave for 15 min at 121oC, andpour into sterile Petri dishes.

TY Agar

Used for the cultivation of Pseudomonas and Rhizobium.

Composition (g/L):

Tryptone 5.0 g

Yeast Extract 3.0 gCaCl2 0.5 g

MgSO4 0.1 gAgar 13.0 g

Add distilled water to a final volume of 1 L, autoclave for 15 min. at 121 oC, and pour

into sterile Petri dishes.

Luria Methylene Blue Agar

Used for the observation of coliphage plaques.

Composition (g/L):

Tryptone 10.0 gYeast Extract 5.0 g

NaCl 5.0 gGlucose 1.0 g

Methylene Blue 0.02 gAgar 15.0 g

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Dissolve in distilled deionised H2O to a final volume of 1 L, autoclave for 20 minutes at15 psi (1.05 kg/cm2) on liquid cycle, and pour into sterile Petri dishes.

Luria Agar Overlay

Used for the propagation of coliphage.

Composition (g/L):

Tryptone 10.0 g

NaCl 5.0 gGlucose 1.0 g

CaCl2 0.11 gAgar 6.0 g

Add 3 mL of NaOH per L and check for a pH of 7.2. Add agar, dissolve, then autoclave

for 20 minutes at 15 psi (1.05 kg/cm2) on liquid cycle.

Peptone Yeast Extract Medium (PYE)

Used for the propagation of soil and compost microorganisms.

Composition (g/L)

Peptone 10.0 gNaCl 5.0 g

Yeast Extract 5.0 gFor solid medium:

Agar 15.0 g

Dissolve in distilled deionised H2O to a final volume of 1 L, autoclave for 20 minutes at15 psi (1.05 kg/cm2) on liquid cycle, and pour into sterile Petri dishes.

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Yeast Peptone Dextrose Medium (YPD; YEPD)

YPD is a complex medium used for yeast.

Composition (g/L):

Yeast extract 10.0 gPeptone 20.0 g

Glucose 20.0 gFor solid medium:

Agar 15.0 g

Dissolve in distilled deionised H2O to a final volume of 1 L, autoclave for 20 minutes at15 psi (1.05 kg/cm2) on liquid cycle, and pour into sterile Petri dishes.

Hugh and Leifson’s Medium

Used for detection of glucose or lactose degradation by microorganisms.

Composition (g/L):

Peptone from meat 2.0 g

NaCl 5.0 gK2HPO4 0.3 g

Carbohydrate 10.0 gAgar 5.0 g

Bromothymol blue 3.0 mL of a 1% solution

Bromothymol blue is dissolved in water. Alcoholic solutions of indicators should not beused as acid may be produced from the alcohol. For critical studies, the carbohydrate

should be sterilised completely and added to the otherwise complete sterile medium.

Autoclave 20 minutes on liquid cycle (121 oC; 15 psi). Dispense into sterile test tubes to afinal volume of 5 mL per tube. Note, could be dissolved by heating first, dispensed, then

the tubes autoclave to sterilise.

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Eosin Methylene Blue Agar:

Used for selection of Gram negative bacteria, and differentiation of lactose fermentingorganisms.

Composition (g/L):

Peptones 10.0 g

Di-potassium hydrogen phosphate 2.0 gLactose 5.0 g

Sucrose 5.0 gEosin Y, yellowish 0.4 g

Methylene blue 0.07 gAgar 15 g

Dissolve in distilled water to a final volume of 1 L, autoclave 15 min at 121oC, and pour

plates.

MacConkey Agar:

Used for selection of Gram negative bacteria, and differentiation of lactose fermentingorganisms.

Composition (g/L):

Peptone 20.0 g

NaCl 5.0 gLactose 10.0 g

Bile salts 5.0 gNeutral red 0.075 g

Agar 12.0 g

Dissolve in distilled water to a final volume of 1 L, autoclave 15 min at 121oC, and pourplates.

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Casein Agar:

Used for the determination of caseolytic activity of microorganisms.

Composition (g/L):

Agar 10.0 gSkim milk 100.0 g

Add distilled water to a final volume of 1 L, autoclave for 15 min at 121 oC and pour into

sterile Petri dishes.

Indole Broth:

Used for the differentiation of organisms that can metabolise tryptophan to produceindole.

Composition (g/L):

K2HPO4 15.65g

L-Tryptophan 5.0 gNaCl 5.0 g

KH2PO4 1.35g

Dissolve in distilled water to a final volume of 1 L, dispense into test tubes, and autoclave15 min at 121oC.

Methyl Red Voges Proskauer (MRVP) Broth:

Used for the differentiation of bacteria based on acid production (methyl red test) or

acetoin production (Voges Proskauer reaction).

Composition (g/L):

Glucose: 5.0 gKH2PO4 5.0 g

Pancreatic digest of casein 3.5 gPeptic digest of animal tissue 3.5 g

Dissolve in distilled water to a final volume of 1 L, dispense into test tubes, and autoclave

15 min at 121oC.

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Simmons Citrate Agar:

Used for the detection of citrate degradation by microbes. Organisms able to utilisecitrate exhibit growth and the medium changes from green to blue. Citrate negative

organisms do not grow and the medium remains green.

Composition (g/L):

Ammonium dihydrogen phosphate 1.0 gPotassium dihydrogen phosphate 1.0 g

NaCl 5.0 gSodium citrate 2.0 g

Magnesium sulphate 0.2 gBromthymol blue 0.08 g

Agar 15.0 g

Dissolve in distilled water to a final volume of 1 L, dispense into test tubes, autoclave 15

min at 121oC, and cool in the slant position.

Urea Broth:

Urea broth supports the growth of microorganisms that can utilise urea as their solecarbon source. Organisms that are able to metabolise urea change the incorporated

indicator to red.

Composition (g/L):

Yeast extract 0.1 gKH2PO4 9.1 g

Disodium hydrogen phosphate 9.5 gUrea 20.0 g

Phenol red 0.01g

Dissolve in distilled water to a final volume of 1 L, heating to 60oC if neccessary. Steriliseby filtration and dispense into sterile test-tubes. Do not autoclave.

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Sucrose Agar:

Used for differentiation of bacteria based on their ability to produce dextran – a polymerof sucrose.

Composition (g/L):

Beef Heart (solids from infusion) 500.0 g

Sucrose 50.0 gAgar 20.0 g

Tryptose 10.0 gNaCl 5.0 g

Dissolve in distilled water to a final volume of 1 L, autoclave for 15 min at 121oC, and

pour into sterile Petri dishes.

Litmus Milk:

Used for the maintenance of lactic acid bacteria and the differentiation of bacteria based

on their action in milk.

Composition (g/L):

Skim milk 100.0 gAzolitmin 0.5 g

Na2SO3 0.5 g

Dissolve in distilled water to a final volume of 1 L, heat to boiling and dispense into testtubes. Autoclave for 20 min at 115oC.

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Motility Medium S:

Used for the determination of bacterial motility.

Composition (g/L):

Beef heart solids from infusion 500.0 gGelatin 30.0 g

Enzymatic hydrolyzate of protein 10.0gNaCl 5.0 g

K2HPO4 2.0 gKNO3 2.0 g

Agar 1.0 g2,3,5-triphenyltetrazolium chloride solution* 10 mL

Add all components except triphenyltetrazolium chloride solution to distilled water;

bring volume to 990 mL. Mix thoroughly, and heat to boiling. Autoclave for 15 min. at121oC, let cool to 60oC and add 10 mL of triphenyltetrazolium chloride. Dispense into

sterile test tubes.

* To prepare 2,3,5-triphenyltetrazolium chloride solution, dissolve 0.1 g in 10 ml ofdistilled water. Mix thoroughly and filter sterilise.

References:

Atlas, R.M., and Parks, L.C. 1993. Handbook of Microbiological Media. CRC Press, Inc.Boca Raton, Florida.

Difco Manual: Dehydrated Culture Media and Reagents for Microbiology. 10th Ed.(1984). Difco Laboratories, Detroit, Michigan.

Merck Microbiology Manual 1994. Merck, Darmstadt, Germany.

Ross, H. 1992/3. Microbiology 241 Lab Manual. University of Calgary Press, Calgary.

Sambrook, J. and Russell, D. W. 2001. Molecular Cloning – A Laboratory Manual. 3rd

edition. Cold Spring Harbor Laboratory Press, New York.

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REAGENTS:

Ethanol, 70%:

95% Ethanol 36.8 mL

Distilled Water 13.2 mL

Barritt’s Reagents:

Solution A: Dissolve 6 g alpha naphthol in 100 mL 95% ethanolSolution B: Dissolve 16 g potassium hydroxide in 100 mL distilled water.

Crystal Violet Stain:

Solution A: Dissolve 2.0 g of crystal violet in 20 mL of 95% ethanol.

Solution B: Dissolve 0.8 g of ammonium oxalate in 80 mL of distilled water.Mix solutions A and B.

Gram’s Iodine:

Dissolve 2 g of potassium iodide in 300 mL of distilled water; then add 1 g of iodine

crystals.

Kovac’s Reagent:

Mix the following:n-Amyl alcohol 75 mL

Hydrochloric acid 25 mLp-dimethylamine-benzaldehyde 5.0 g

Malachite Green Stain:

Dissolve 5 g of malachite green oxalate in 100 mL of distilled water.

Nigrosin Solution:

Add 10 g of nigrosin (water soluble) to 100 mL of distilled water. Boil for 30 min, and

add 0.5 mL of formaldehyde (40%). Filter twice through double filter paper. Store underaseptic conditions.

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Oxidase Test Reagent:

Dissolve 1 g of dimethyl-p-phenylenediamine hydrochloride in 100 mL of distilled water.Make fresh.

Phloxine B:

Dissolve 1 g of phloxine in 100 mL of distilled water.

Safranin:

Dissolve 0.25g safranin in 10 mL of 95% ethanol. Add to 100 mL of distilled water.

Sudan Black Stain:

Dissolve 0.3 g of Sudan Black in 100 mL of 70% ethanol. Shake before each use.

References:

Clark, G. (1984) Staining Procedures. 4th Ed. Williams and Wilkins, Baltimore, Maryland.

Benson, H.J. (1985). Microbiological Applications: A Laboratory Manual in GeneralMicrobiology, 4th Ed. Wm. C. Brown Publishers, Dubuque, Iowa.

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pH INDICATORS:

Table 1: Indicators of Hydrogen Ion Concentration.

pH Indicator pH Range Full Acid Colour Full AlkalineC lCresol Red 0.2 - 0.8 Red Yellow

Meta Cresol Purple(acid range)

1.2 - 2.8 Red Yellow

Thymol Blue 1.2 - 2.8 Red Yellow

Brom Phenol Blue 3.0 - 4.6 Yellow Blue

Brom Cresol Green 3.8 - 5.4 Yellow Blue

Chlor Cresol Green 4.0 - 5.6 Yellow Blue

Methyl Red 4.4 - 6.4 Red Yellow

Chlor Phenol Red 4.8 - 6.4 Yellow Red

Brom Cresol Purple 5.2 - 6.8 Yellow Purple

Bromothymol Blue 6.0 - 7.6 Yellow Blue

Neutral Red 6.8 - 8.0 Red Amber

Phenol Red 6.8 - 8.4 Yellow Red

Cresol Red 7.2 - 8.8 Yellow Red

Meta Cresol Purple(alkaline range)

7.4 - 9.0 Yellow Purple

Thymol Blue(alkaline range)

8.0 - 9.6 Yellow Blue

Cresolphthalein 8.2 - 9.8 Colourless Red

Phenolphthalein 8.3 - 10.0 Colourless Red

Adapted from: Benson, H.J. (1985). Microbiological Applications: A Laboratory Manual inGeneral Microbiology, 4th Ed. Wm. C. Brown Publishers, Dubuque, Iowa.

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APPENDIX 9Care and Feeding of the Microscopes

Checklist For Compound MicroscopesName:________________________________________Class and section: ______________________________Date:_________________________________________Microscope #: _________________________________Did you find the microscope in proper workingorder? Y or N If not, what was the problem?______________________________________________________________________________________________________________________________________________________________________________________ Slide removed from stage__ Slide, stage, and objectives are free of oil__ Mechanical stage is centered__ Stage placed at its lowest position__ 4x objective placed into working position__ Ocular micrometer replaced with regular ocular__ Binocular head secured in “start” position__ Rheostat turned to 0 and lamp is turned off__ Cord is wrapped tightly around arm and lamp__ Cord is secured with cord clip__ Dust cover is placed over scope

Checklist For Dissecting ScopesName:________________________________________Class and section: ______________________________Date:_________________________________________Microscope #: _________________________________Did you find the microscope in proper workingorder? Y or N If not, what was the problem?______________________________________________________________________________________________________________________________________________________________________________________ Turn off transformer__ Unplug transformer and lamp__ Wrap cord tightly around transformer__ Place transformer on stage with binocular head well abovethe transformer__ Replace dust cover