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MICRO 6TOOLS OF THE LABORATORY

most habitats harbor microbes in complex associations often necessary to separate microbes in order to

identify and study them – need to be grown in artificial conditions

need an oil immersion microscope to view

procedures for investigating and characterizing microorganisms – the six I’s Inoculation Incubation Isolation Inspection Information gathering identification

METHODS OF MICROBIAL INVESTIGATION

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sample the object of interest (almost anything)

Common sources Bodily fluids

Foods

Water

Soil

Plants

Animals

Icebergs

Volcanoes

Rocks

SPECIMEN COLLECTION

sample is placed on a medium that will support its growth (human biological samples – blood agar plate)

medium may be solid or l iquid – held in tubes, plates, flasks and even eggs

sample delivery tool is usually a loop, needle, swab or syringe

INOCULATION

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inoculated media are placed in a controlled environment (incubator) to promote growth (body temp / aerobic or anaerobic)

culture of visible colonies develop on the media

INCUBATION

Inoculation technique that separates microbes into distinct colonies that contain a single type of microbe – want a pure culture

ISOLATION

Throat culture –alpha – hemolytic colonies (Strep)

Urine culture – pink colonies on McConkeyGram stain – GNR (E coli)

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cultures are observed for macroscopic appearance of growth characteristics

samples of the culture are stained and viewed under the microscope – cell type and shape

INSPECTION

Identify the microorganism using info gathered from inspection and investigation

accomplished through keys, charts and computer programs that analyze data

IDENTIFICATION

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optical microscopy

sample is mounted on a glass slide that sits on the stage between the condenser and the objective lens

the manner in which the specimen (mount) is prepared depends upon the condition of the specimen (living or preserved)

aim of the examiner – observe overall structure, identify microorganisms or see movement

type of microscopy available – bright field,

dark field, phase contrast or fluorescence

PREPARING SPECIMENS FOR OPTICAL MICROSCOPES

l ive samples – wet mounts – microbes can be observed as close to their natural state as possible

cells are suspended in a fluid (water, broth, saline) –temporarily maintains viabil ity and provides a medium to see locomotion a drop of liquid culture placed between a glass slide and a

coverslip (glass cover)

FRESH LIVING PREPS

Trichomonas vaginalis

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hanging drop slide – special concave (depression) slide, an adhesive (sealant) and coverslip from which a small drop of sample is suspended

short term mount – true assessment of the size, shape, arrangement, color and motility of cells

sreater cellular detail with phase-contrast or interference microscope

Trichomonas in urine samples

permanent mount for long-term study – fixed, stained specimen

smear technique (Robert Koch)

spread a thin film of specimen on a slide and air dry

heat fixation (heat gently) – simultaneously kills the specimen and secures it to the slide – preserves cellular components

stain for cell differentiation

FIXED, STAINED SMEARS

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dyes impart color to cells by affixing to them through chemical reactions

basic (cationic) dyes – positive charge

acidic (anionic) dyes – negative charge

Negative / Positive Staining most procedures involve positive staining – dye sticks to

cells and gives them color

negative stain – dye does not stick to specimen but dries around its outer boundary (silhouette) – cells do not stain b/c negative stain is repelled by negative charge of cell surface

smear is not fixed – simple stain to show cellular shape, size, arrangement and accentuate capsules

Nigrosin (blue / black)

India ink (black suspension of carbon

particles)

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Simple vs Differential Staining

positive staining methods are classified as simple, differential or structural

simple stains – require only a single dye that readily bind to bacterial cells (malachite green, crystal violet, basic fuchsin, safranin, methylene blue)

differential stains – require two different colored dyes (primary dye and counterstain) to distinguish between cell types / parts

uses dyes of contrasting color to clearly emphasize differences between cells types / parts (red and purple / red and green/ pink and blue)

show characteristics of size, shape and arrangement

gram stain, acid fast, endospore stains

DIFFERENTIAL STAINS

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GRAM STAIN developed by Has Christian Gram (1884)

two cell reactions: gram-pos (purple) and gram-neg (red)

difference in staining due to structural variations in the cell wall

crystal violet stains all cell purple

Gram’s iodine (mordant (IKI) – key differentiating step) causes the dye to form large crystals that get trapped in the cell wall (b/c cell wall in gm+ cells is thicker, the entrapment of the dye is more extensive than gm- cells)

application of alcohol dissolves lipids in the outer membrane of gm- cells (removes the dye from them)

application of a counterstain (safranin) dyes the colorless gm- cells red

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ACID FAST STAIN important diagnostic stain – differentiates acid fast bacteria

(pink) from non-acid fast bacteria (blue)

originated as a specific method to detect Mycobacterium tuberculosis in specimens

acid fast bacteria have a particularly impervious outer wall that holds fast (tightly / tenaciously) to carbol fuchsin (dye) – even when washed with a solution containing acid or acid alcohol

ENDOSPORE STAIN

similar to the acid fast method – dye is forced by heat into resistant survival cells (spores / endospores)

designed to distinguish between spores and the vegetative cells that make them

medically significant microbes are gm+ spore-forming rods (Bacillus - botulism and Clostridium - tetanus)

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used to emphasize special cell parts – capsules, endospores, and flagella that are not revealed with conventional staining

capsule staining – capsule does not react with most stains – usually negatively stained with India Ink – differential in that not all microbes exhibit capsules (Cryptococcus)

flagellar staining – flagella are enlarged by depositing a coating on the outside of the fi lament and then staining –presence and number of flagella helpful in bacterial differentiation

STRUCTURAL STAINS

PART 2

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inoculation (to culture bacteria) - a tiny amount of sample (inoculum) into medium (media) which provides an environment for multiplication

culture – the observable growth on the media

the nature of the sample being cultured depends on the objectives of the analysis

clinical specimens for determining cause of infectious disease – obtained from body fluids (blood, CSF) discharges (urine, feces, sputum) or diseased tissue (wounds, burns, ulcers)

other samples for analysis can include nearly any natural material (soil, water, sewage, foods, air and inanimate objects)

INOCULATION, GROWTH AND IDENTIFICATION OF CULTURES

Special Requirements of Culturing

Sterile (complete absence of viable microbes), aseptic(prevention of disease) and pure culture (growth of a single culture) techniques

contamination during inoculation is a constant problem –sterile techniques (media, transfer equipment) help ensure that only microbes from the sample are cultured

concern – possible release of infectious agents from cultures into the environment – prevented by aseptic technique (keeping species in a pure culture for further study, ID or biotechnology applications

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based on the concept that if an individual bacterial cell is separated from other cells and provided with adequate space on a nutrient surface, it wil l grow into a discrete mound of cells (colony)

colony consists of one species – arises from a single cell or a small cluster of cells

ISOLATION TECHNIQUES

based on the concept that if an individual bacterial cell is separated from other cells and provided with adequate space on a nutrient surface, it wil l grow into a discrete mound of cells (colony)

colony consists of one species – arises from a single cell or a small cluster of cells

ISOLATION TECHNIQUES

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streak plate method mall droplet / amount of sample is spread with an inoculating loop over the surface of the medium in a pattern that gradually thins out the sample – separates cells spatially over several sections of the plate

loop dilution or pour plate technique sample is inoculated into a series of cooled but sti l l l iquid

agar tubes so as to dilute the number of cells in each successive tube in the series

inoculated tubes are plated (poured) out in to sterile petri dishes and allowed to solidify (harden)

some colonies may develop deep in the medium on not just on the surface

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spread plate technique small volume of l iquid from a diluted sample is pipetted onto

the surface of the medium and spread around evenly by a sterile spreading tool (hockey stick, glass spreader)

as with the streak plate, cells are spread over separate areas on the surface to form individual colonies

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once the medium has been inoculated, it is incubated in a temp-controlled chamber (incubator) to encourage microbial growth

usual temps used in the clinical lab fall between 20-40°C

Incubators can also control the content of atmosphere gases (O2 / CO2) required by some bacterial pathogens

Incubation period ranges from a few hours to several weeks

culture multiples to produce macroscopically visible colonies

pure culture (axenic) – culture free of other l iving things except for the one being studied – only one species of bacteria

used to identify pathogenic bacteria in a lab setting

mixed culture – contains two or more easily differentiated species of bacteria

may contain contaminants – species other than the pathogen

subculture – method of taking a

small sample of a distinct colony of

bacteria and transferring it to new

media to obtain a pure culture

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gram stain colony growth on differential media

biochemical tests – determine fundamental chemical characteristics, products produced during growth, presence of enzymes, and mechanisms for deriving energy

immunologic testing – test isolate against antibodies animal studies DNA profiles

IDENTIFICATION TECHNIQUES

traditional pathway in bacterial identification uses flowcharts or keys that apply the results of tests to a selection process (points of separation are based on a positive or negative result)

this process of “keying out” the microorganism can simplify the identification process

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PART 3

some microbes require only a few simple organic nutrients for growth – others need a complex list of specific compounds

at least 500 types of media used in culturing and identifying microorganisms

media contained in tubes, flasks, or petri dishes –inoculated with tools l ike loops, needles, pipettes and swabs

media are varied in nutrient content, consistency and can be formulated for a specific purpose – sterile technique imperative

MEDIA THE FOUNDATIONS OF CULTURING

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LIQUID MEDIA

water-based solutions that do not solidify at temps above freezing and that tend to flow freely when the container is ti lted

termed broths, milks or infusions – nutrient solutes are dissolved in water

growth occurs throughout the container – dispersed, cloudy or flakey appearance

common lab medium – nutrient broth – beef extract and peptone (amino acid) in sterile water

methylene blue milk / litmus milk (whole milk and dyes)

fluid thioglycollate (slighlty viscous broth used to determine patterns of growth in O2)

PHYSICAL STATES OF MEDIA

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SEMISOLID MEDIA

clot-l ike consistency at room temp – contain a solidifying agent (agar / gelatin) that thickens but does not solidify

used to determine the motil ity of bacteria and to localize a rxnat a specific site

Motil it iy test medium / sulfur indole motil ity (SIM) medium

contain a small amount of agar

(0.3-0.5%) – medium is stabbed

carefully in the center

(inoculating needle) – observed for

pattern of growth around the stab line

SOLID MEDIA

provide a firm surface on which cells can form discrete colonies – advantageous for isolating and culturing bacteria and viruses

two forms

liquefiable solid media (reversible solid media – changes its physical properties in response to temp) – agar(polysaccharide) is flexible, moldable and provides a basic matrix to hold moisture and nutrients – agar is not digestible (not a nutrient for microorganisms) nutrient agar (beef extract, peptone and 1.5% agar)

non-liquefiable solid media – do not melt and include material like rice grains (used to grow fungi), cooked meat media (anaerobes), and egg / serum media (permanently coagulated/ hardened by moist heat)

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Pseudomonas on nutrient agar /

blood agar

Plant fungi on solid agar

media with chemically defined composition – synthetic –contain pure chemical nutrients that vary l itt le from one source to another and have a molecular content specified by a formula

media that cannot be defined by an exact chemical formula –nonsynthetic or complex media

supplements / extracts from animal or plant tissues – ground up cells / secretions, blood , serum, meat extracts, infusions, milk, soybean digests, peptone (partially digested protein rich in amino acids (aa) – used as a C and N source)

CHEMICAL CONTENT OF MEDIA

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General Purpose Media

designed to grow a broad spectrum of microbes that do not have special growth requirements

Nonsynthetic (complex) and contain a mix of nutrients that can support a variety of bacteria and fungi

nutrient agar / broth, brain-heart infusion, trypticase soy agar (TSA – contains partially digested mil protein (casein), soybean digest, NaCl and agar)

Enriched Media

contains complex organic substances – blood, serum, hemoglobin, or special growth factors (vitamins, amino acids that bacteria cannot synthesize) that certain species need in order to grow

fastidious bacteria – require growth factors and complex nutrients (blood agar / chocolate agar)

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SELECTIVE AND DIFFERENTIAL MEDIA

• designed for special microbial groups

• can provide a preliminary ID of a genus / species in a single step

selective medium

• contains one or more agents that inhibit the growth of certain microbes – selects a microbe and allows it to grow by itself

• important in the primary isolation of a specific type of organism from samples containing a mixture of organisms (feces, urine, saliva, skin, water, soil) – suppress the unwanted organisms and allow growth of the desired ones

MEDIUM SELECTIVE AGENT USED FOR…

Mannitol salt agar 7.5% NaCl Isolation S Aureus from infections material

Enterococcus faecalis broth sodium azide, tetrazolium Isolation of fecal Enterococci

Phenylethanol agar (PEA) phenylethanol chloride Isolation of Staph / Strep

Tomato juice agar Tomato juice, acid Isolation of lactobacilli from saliva

MacConkey agar (MAC) Bile, crystal violet Isolation of gram negative enterics

Eoisin-methylene blue (EMB) Bile, dyes Isolation of coliform bacteria

Salmonella/Shigella (SS) Bile, citrate, brilliant green Isolation of Salmonella and Shigella

Sabouraud’s agar (SAB) pH 5.6 (acid) inhibits bacteria Isolation of fungi

Examples of Selective Media, Agents and FunctionsTable 3.7 (p-81 textbook)

• make sure you have in notes

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differential media

• grow several types of microorganisms but are designed to bring out visible differences among those organisms – colony size and color, formation of gas bubbles or precipitates

• variations come from the types of chemicals contained in the media and the ways the microbes react to them

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MEDIUM Substances that facilitate differentiation Differentiates

Blood agar (BAP) Intact RBC Types of hemolysis (RBC damage)

Mannitol Salt Agar (MSA) Mannitol and phenol red Species of Staphylococcus

Hektoen enteric (HE) agar Brom thymol blue, acid fuchsin, sucrose, salicin, thiosulfate, ferric ammonium citrate, bile

Salmonella, Shigella (lactose fermentersfrom nonfermenters – also H2S rxns)

MacConkey agar (MAC) Lactose, neutral red Lactose fermenters (lower pH) from others

Eosin-methylene blue (EMB) Lactose, eosin, methylene blue Same as MAC

Urea broth Urea, phenol red Bacteria hydrolyze urea to ammonia (↑pH)

Sulfur indole motility (SIM) Thiosulfate, iron H2S gas producers, motility, indole formation

Triple-sugar iron (TSIA) Triple sugars, iron, phenol red Sugar fermentation, H2S formation

XLD agar Lysine, xylose, iron, thiosulfate, phenol red Can differentiate: Enterobacter, Escherichia, Proteus, Providencia, Salmonella and Shigella

Examples of Differential Media (Table 3.8 / p-82 text)

• make sure you put in notes