Partial Materials in the Final Lab Exam (Nov. 28/29): Labs #9-23 (All labs after the first lab exam)

Please also read the review sheets I handed out on Nov. 20 in lab. I will have office hour M/Th: 9:15 a.m. - 10:15 a.m. at DH 553 or DH 543 (my lab)

LAB 9: Single Colony Isolation

Know how to obtain single colonies through the “Streak for Isolation” on an agar plate technique.

Streak Plate technique

Klebsiella roseaSerratia marcescens

All in NA

plates

Lab #9

Micrococcus luteus Staphylococcus aureus

Blood agar (BA) is a differential medium. - Some bacteria produce an enzyme that is able to lyse RBCs – this process is hemolysis. - By growing bacteria on blood agar we can determine if the

bacteria produce hemolysin and thus lyse the RBCs. - Blood agar is NA to which sheep RBCs have been added. - If hemolysin is produced by the bacteria it will be secreted

into the medium and the RBCs will be lysed (the medium will be clear rather than red).

- So presence of clearing around the bacterial growth indicates hemolysis.

- Growth on BA differentiates between the hemolytic and non-hemolytic bacteria.

Gamma hemolysis = No hemolysisAlpha hemolysis = PartialBeta hemolysis = Complete

Lab 10: Stock and Working Slants

• Why did we prepare a stock and a working stock slant for the unknown?

• Why did we grow the unknown in different media and under different conditions?

“Working” “Stock”

Lab 11: Simple Staining & Bacterial Smear

• Understand simple, negative, and positive staining.• Know how to prepare a bacteria smear

Demos: simple stains of: Neisseria (diplococci) Pseudomonas (bacilli)

• Know the entire Gram Staining procedure and the function of each step.

• Know the Endospore procedure (in Appendix, p. 121)

Lab 12: Differential Staining (Gram Stain)

GRAM STAIN

E coli (Gm -) Staph epidermidis (Gm+)

ENDOSPORE STAIN

Bacillus megaterium

Bacillus anthracis

Clostridium tetani

See Appendix IV, p. 121Outcome for endospore + for Micr20

Cell Arrangements:

Lab 13: Selective and Differential Media

• EMB: Eosin-Methylene Bluea. Differential and selective properties.

b.Contains bile salts and the dyes eosin and methylene blue; all inhibitory to Gram-positive bacteria (e.g. Staphylococcus aureus).

c. Selects for Gram-negative bacteria (e.g. Escherichia. coli).

d.Differentiates lactose fermenting (dark color with metallic sheen) from non-lactose-fermenting (colorless) bacteria.

Salmonella pullorum

Staph. epidermidis

E. coli

Staph. aureus

TGA: Tellurite Glycine Agara.Selects for coagulase-positive staphylococci.

b.Differential: coagulase-positive cocci form black colonies.

c.Coagulase-negative cocci are generally inhibited. The ones that grow are gray.

d.Most Bacilli and Pseudomonas (Gm+) are inhibited.

e.Proteus sp rarely grows and form brown colonies.

Lab 13: Selective and Differential Media

E. coli

Staph. epidermidis

Staph. aureus

Salmonella pullorum

Know all the media we covered in Micro20 since lab #9: The purpose of the medium, how to read a positive and a negative result, what those results mean, and the MAJOR components of the medium.

Lab 14: Antibiotic Sensitivity

• Antibiotics are chemicals that are produced by other bacteria/fungi that have the ability to prevent other organisms (bacteria) from growing or killing them.

• Sensitivity X Resistance to antibiotics.

• Bacteriostasis (stopping bacterial growth) X bacteriocide (killing of bacteria).• Broad spectrum antibiotics- effective against a wide range of bacteria (G+ and G-).• Narrow spectrum antibiotics - effective against a small specific group of bacteria (either G+ or G-).

Lab 14: Antibiotic Sensitivity; Disc Diffusion Method

LAB 15: Catalase, Amylase, Gelatinase (Proteinase), MRVP

• MRVP, see Appendix IV, p.118-119

CATALASE

Negative Positive

Streptococcus aureus

H2O2

Enterococcus faecalis

Amylase: Starch Hydrolysis

BEFORE AFTER

E coli - (neg.)Bacillus subtilis + (pos.)

Flood with Iodine solutionE.coli

Bacillus subtilis

Gelatinase test: Plate was flooded with Frazier’s Developer

Gelatinase +

Gelatinase negative

LAB 16: Urease, SIM agar, Citrate

UREASE• UREASE TEST: Urease is an enzyme that

breaks the carbon-nitrogen bond of amides (e.g. urea) to form carbon dioxide, ammonia, and water. Members of genus Proteus are known to produce urease. When urea is broken down, ammonia is released and the pH of the medium increases (becomes more basic). This pH change is detected by a pH indicator that turns pink in a basic environment. A pink medium indicates a positive test for urease.

SIM agar• SIM = Sulfide, Indole, Motility.

• INDOLE TEST: Indole is a component of the amino acid tryptophan. Some bacteria have the ability to break down tryptophan for nutritional needs using the enzyme tryptophanase. When tryptophan is broken down, the presence of indole can be detected through the use of Kovacs' reagent. Kovac's reagent, which is yellow, reacts with indole and produces a red color on the surface of the test tube.

SIM agar

MOTILITY

Non-motile bacterium

(e.g. Staph aureus)

Motile bacterium

(e.g. Pseudomonas aeruginosa)

Citrate: The Simmon’s Citrate medium tests the ability of the bacteria culture to be able to use citrate as the sole C source.

Bacteria that are able to produce the enzyme citrase are able to transport the citrate into the cell and use it as a source of C.

Since the medium does not contain any other source for C, only those bacteria that can produce citrase are able to grow in this medium.

When cultures are able to use the citrate they break it down, producing sodium bicarbonate, which changes the pH of themedium to alkaline.

The pH indicator in the medium (bromothymol blue) changes to a blue color from its original green color. +

LAB 17: Carbohydrate Utilization

Lab 17: KIA medium• C = Uninoculated 3 = Glucose fermenter + H2S producer• 1 = Non-fermenter 4 = Glucose+Lactose fermenter, gas• 2 = Glucose fermenter 5 = Gluc + Lact ferm + H2S producer

Lab 17: Fermentation of Carbohydrates

F- tubes

SUGAR FermentationDetection is based on acid production due to sugar fermentation. The pH indicator is PHENOL RED. Phenol red turns yellow under acidic conditions. Hence, yellow means a positive result.

The ability to ferment specific sugars is dependant on the ability of the bacterium to produce the specific enzymes required for the transport and metabolism of that particular sugar.

Thus fermentation of various sugars can be used to characterize bacteria.The F-tubes use phenol red in the medium as pH indicator and the use

of inverted tubes to detect production of gases.

Results are recorded as Negative (no metabolism); Acid (+ reaction);Acid + Gas (+ with gas production).

LAB 18: Unknown & Single Colony Isolation

Know how to use the Dichotomous Key to identify a bacterium based on morphology, Gram staining, endospore production, and various metabolic reactions. See p.58-61of lab manual.

LAB 19: Pour Plate

•Pour Plate Technique•Serial Dilution•Colony Forming Unit (CFU)•Quantification of Bacteria in Cell/ml

Lab 19: Pour Plate

• Bacteria Enumeration

Dilution Series:

100 10-1 10-2 10-3 10-4 (Dilution) (1:1 1:10 1:100 1:1000 1:10,000)

TMTC 1000 400 100* 20 (CFU)

*Best 30-300 CFU•CFU = 100• Dilution = 1000•Hence 100 X 1,000 =100,000 = 1x105

1ml 1ml 1ml 1ml

1 2 3 4 5

Bacteria Enumeration

1x10-5 1x10-6

Cell /ml= (CFU X dilution factor) / volume

LAB 20: Most Probable Number

• MPN method, MPN table• Durham tubes• Presumptive, Confirmed, and Completed tests

Lab 20: Most Probable Number (MPN)

Bacteria Enumeration (Presumptive)

1st- Presumptive test: growth on lauryl tryptose broth

2nd - Confirmed test: on Eosine-Methylene Blue Agar (EMB)

3rd - Completed tests

MPN method:

LAB 21: Phage Characterization and Quantification

• Plaque, Plaque Forming Unit (PFU)• Serial Dilution, Phage quantification• T1phage

BACTERIOPHAGE

1x10-4 Dilution

1x10-6 Dilution

Plaque(clear zone)

LAB 22: Bacterial Aggutination & Immunoprecipitation

•

Immunoprecipitation- is the reaction between a soluble antigen and its specific antibodies-soluble antigens are smaller and in solution; complexing with antibodies make these

bigger and they fall out of solution as a precipitate –visible to the eye.

Antibody specificity known (toxin, protein, etc.)Antigen presence or identity not known (?)Precipitationreaction between antibody and soluble antigen

Immunoprecipitation

Antibody specificity known (toxin, protein, etc.)Antigen presence or identity not known (?)Precipitationreaction between antibody and soluble antigen

Immunoprecipitation

Immunoprecipitation

Reaction of nonidentity

Reaction ofidentity

LAB 23:Staphyloslide Latex Test Kit

• Example of Agglutination

Bacterial agglutination

S. aureus S. epidermidis

?

agglutination no agglutination

S. aureus S. epidermidis

Bacterial agglutination (new procedure).

#1Mark your bacterial agglutination cards

S. aureus S. epidermidis

Bacterial agglutination

#2Mix the latex agglutination reagent dropper bottle and dispense one drop onto each circle

Bacterial agglutination

#3Using a sterile toothpick , pick up and smear 1 suspect colony from your negative control in the proper ring.

#4Using a NEW sterile toothpick , pick up and smear 1 suspect colony from your positive control in the proper ring.

#5Using a NEW sterile toothpick , pick up and smear 1 suspect colony from your unknown in the proper ring.

S. aureus S. epidermidis

S. aureus S. epidermidis

Bacterial agglutination (Part B).

#6Pick up and gently rock the card for 20 seconds and observe for agglutination under normal lighting conditions

?Bacterial antigen: Agglutination with anti-S. aureus :

S. aureus

S. epidermidis

unknown