MICROBIOLOGICAL METHODS

5 BASIC TECHNIQUES

INOCULATION INCUBATION

INSPECTION ISOLATION

IDENTIFICATION

INOCULATION

Introduction of small sample of cells (INOCLUM) into a container of nutrient medium

CLINICAL SAMPLE - blood, urine , CSF, feces, etc

HABITAT SMAPLE - soil, water, sewage, food, etc

CONTAINERS

(individual) test tube, flask, agar plate (petri dish)

(industry) large scale fermenters

MEDIA

provides nutritional requirements for organisms

SIMPLE - few inorganic compounds

COMPLEX - inorganic & organic compounds

PHYSICALSTATE

CHEMICALCOMPOSITION

FUNCTIONALTYPE (Purpose)

Liquid Synthetic(Chemically)

General Purpose

Semi-solid Enriched

Solid (Liquid) Selective

Solid Non-synthetic(not chemical)

Differential

AnaerobicGrowth

Specimentransport

Assay

Enumeration

LIQUID MEDIA

Water based solutions, do not solidify at temps above freezing, flow freely in containers

BROTHS, MILKS, INFUSIONS

various solutes dissolved in distilled water

SEMI SOLID MEDIA

clot like consistency, contain solidifying agent (agar/gelatin - 0.3-0.5%)

Used to determine motility, localize reaction at specific sites

SOLID MEDIA

firm surface, allows cells to form discrete colonies

Advantageous for ISOLATION/SUBCULTURING

2 Forms:

LIQUEFIABLE : reversible solid, agar, thermoplastic

NON LIQUEFIABLE :NOT thermoplastic, cooked meat, potato slices, egg media

THERMOPLASTIC - solid at RTP/incubation temps

liquid at 100oC resolidifies at 42oC

CHEMICAL CONTENT

SYNTHETIC - Chemically defined media

Highly pure organic & inorganic compounds

COMPLEX - (Non synthetic) - one ingredient

not chemically definable

Of plant, animal or yeast extract

GENERAL PURPOSE MEDIA

Used for a broad spectrum of microbes, non synthetic

Examples: Brain-heart infusion

Tryptose soy agar

Tryptose soy broth

ENRICHMENT MEDIUM

complex organic substances : blood, serum, growth factors

Used for FASTIDIOUS ORGANISMS

Streptococcus pneumoniae

Requires blood - sterile horse, sheep or rabbit

SELECTIVE & DIFFERENTIAL MEDIA

designed for isolation & identification of specific groups of microbes from mixed populations

SELECTIVE - contains 1 or more inhibitory agents

DYES, ACID, ANTIMICROBIAL AGENTS

Example: growth of A, B and C INHIBITED, but selective growth of D

Examples:

MANNITOL SALT AGAR - 7.5% NaCl, inhibitory [ ] to human pathogen’s

MAcCONKEY AGAR/DEOXYCHOLATE CITRATE AGAR - High Bile salt [ ], inhibitory to Gram +ve bacteria

SABOURAUD’S AGAR (Fungi) - pH 5.6 (acid), inhibits bacteria

DIFFERENTIAL MEDIUM

allows for growth of several types

BUT highlights differences

Colony size, colour, formation of gas, ppt

DYES (differential agents) - act as pH indicators

colour change due to production of acid or base

EXAMPLES

MAcCONKEY AGAR - lactose + neutral red

E. coli produces acid, metabolizes lactose

RED-PINK colonies

Salmonella sp produce no acid

OFF WHITE colonies

E. coli & Salmonella sp. On MacConkey Agar

XYLOSE LYSINE DEOXYCHOLATE AGAR (XLD)

contains xylose, lysine, iron, thiosulphate, bile + phenol red

E.coli acid production

RED-PINK colonies

Salmonella sp convert thiosulphate to H2S gas (SMELL) forms a black ppt with iron

E. coli & Salmonella sp. On XLD Agar

OTHER MEDIA

REDUCING - thioglycollic acid or cystine absorbs oxygen/slows penetration of oxygen

THUS reducing availability

REQUIRED for growing ANAEROBIC BACTERIA

CARBOHYDRATE FERMENTATION - sugars for fermentation, conversion to acids, pH indicator

REQUIRED for BIOCHEMICAL/IDENIFICATION TEST

TRANSPORT - required for maintaining and preserving specimens for a period of time

Examples: STUART’S + AMIES

contains salts, buffers & absorbants

Prevents cell destruction, pH changes, toxic substances NO GROWTH

ASSAY - tests effectiveness of antimicrobial agents, i.e., disinfectants, antiseptics, cosmetics etc.

ENUMERATION - used in industry

allows enumeration of organisms in milk, water, food and soil samples

INCUBATION

Chamber (INCUBATOR)

temperature & atmospheric gas controlled

LAB INCUBATORS : 20 - 40oC

Aerobic or Anaerobic

INCUBATION PERIOD : hours-several weeks depending upon the organism

INSPECTION

Observable growth on or in the medium (CULTURE) at various stages of incubation (EVALUATE GROWTH)

MACROSCOPICALLY - naked eye

LIQUID MEDIA - cloudiness, sediment, scum or colour change

AGAR PLATE - discrete isolated colonies, mass of clinging cells (fungi)

Pseudomonas, Staphylococcus & Serratia on TSA plates

MICROSCOPICALLY individual cells within a colony

Evidence of cellular morphology: size, shape, details of structure

allows for IDENTIFICATION

AIMS

to provide adequate MAGNIFICATION, RESOLUTION and CLARITY of IMAGE

TOTAL POWER OF MAGNIFICATION

Power ofObjective

Power ofOcular

TotalMagnification

40x high (dry) 10x 400x

100x oil imm 10x 1000x

10x lowpower

20x 200x

SUB-CULTURE common microbiological procedure

allows for a pure STOCK-CULTURE of organism

DISPOSAL OF CULTURESmost important - if presents a biological hazard

Autoclaving - steam sterilization

Incineration - burning

Radiation - X or rays

Disinfection - chemical

PREPARATION OF SPECIMENS

MOUNT - a sample on a glass slide

sits between condenser and objective lens

3 FACTORS

1. Condition of specimen (Living or Preserved)

2. Aims of examiner

3. Type of microscope available

LIVING SPECIMENS

Appear as near natural state as possible

Media - suspended in water, broth, saline Allows for motility Temperature - to maintain viability

Advantages: quick & easy to prepare

Disadvantage: no cover slip, susceptible to drying out, free to contamination

FIXED PREPARATIONS

Advantage: Permanent mount, long term study

Smear technique : Developed by Koch >100yrs ago

Disadvantage: KILLS specimen

STAINING PROCEDURES

Any process in which coloured chemicals (DYES) are applied to specimens

DYES - impart colour to cell or cell parts - become affixed through chemical reaction

2 types: BASIC (cationic) +ve charge

ACIDIC (anionic) -ve charge PRINCIPLE : “opposites attract”

EXAMPLES:

BASIC: Crystal violet, methylene blue, safranin

ACIDIC: Nigrosin, india ink

POSITIVE STAINING

+ve stain - sticks to specimen providing colour

Bacillus cereus stained with carbol fuschin (1300x)

NEGATIVE STAINING

-ve stain - (reverse) settles around specimen boundary forms a silhouette (stains the glass slide)

Escherichia coli stained with India ink (1300x)

SIMPLE & DIFFERENTIAL STAINING

+ve staining methods (classification)

Simple - only 1 dye, uncomplicated procedure

Differential - 2 coloured dyes, primary and counterstain, complex procedure

Distinguishes cell types and parts

TYPES OF DIFFERENTIAL STAIN

GRAM’S STAIN - Hans Christian Gram

Differential - colour reaction with cells

Gram +ve bacteria : purple/blue

Gram -ve bacteria : red/pink

Basis for IDENTIFICATION Diagnosis

Gram +ve Staphylococcus aureusGram -ve Escherichia coli

(1400x)

ACID FAST STAIN - Paul Ehrlich

similar to Gram’s, used with resistant bacteria

Acid-fast Bacteria : Pink

Non Acid-fast bacteria : Blue

Mycobacterium : tuberculosis

Mycobacterium tuberculosis (300x)

Mycobacterium marinum

Mycobacterium leprae

ENDOSPORE STAIN

similar to Acid-fast

Distinguishes between bacteria producing spores and those that do not

For Identification of Bacillus sp., Clostridium sp.

Clostridium tetani (1400x)

Gas Gangrene

Anti gas serum - 1934

SPECIAL STAINS

CAPSULE STAIN - specific

undetected by conventional stains

Cryptococcus sp. - fungal infection in AIDS patients

FLAGELLAR STAIN - specific

undetected by microscope due to limited resolving power

Klebsiella pneumoniae

Capsule Stain

BACTERIAL SHAPES

Characteristic Shapes - Bacteria

Spherical - coccoid Cylindrical - rod Spiral - spirilla Pleomorphic - irregularly shaped

BACTERIAL SHAPES

MICROSCOPES

Magnifies size of image

Various types: basic tool

Magnification: enlargement of object

Resolution: degree to which detail is maintained in magnified image

Resolving power: closest spacing between 2 points where can be clearly seen as separate entities

Brightfield Microscope

Extensively used: necessary to view stained specimens

Epifluorescence Microscope

Specimen illuminated at one wavelength of light, observed by light at another wavelength

Uses fluorescent staining

No condenser.

Objective lens focuses light

Useful diagnostic procedures:

Identify microorganisms

Staphylococci in blood - Epifluorescene

Darkfield Microscope

Eliminates need for staining

Achieve contrast between specimen & background

Treponema pallidum (syphilis)

Phase Contrast Microscope

Staining not required

View structures & living organisms

Paramecium caudatum (300x)

Electron Microscopes

Electrons not light beam

Greater resolution

Higher magnifications

Types: Transmission EM

Scanning EM

Transmission EM

Electrons pass through specimen

View ultrastructure of organisms

Influenza virus (360,000x)

Scanning EM

Electron beam scanned across surface of specimen: 3D image

Candida albicans (2200x)

Various Types of Microscopes

TYPE Max Useful Magnification Resolution

Brightfield 1500 x 100-200nm

Darkfield 1500 x 100-200nm

Fluorescence 1500 x 100-200nm

Phase Contrast 1500 x 100-200nm

TEM 500,000 – 1,000,000 x 1-2nm

SEM 10,000 – 1,000,000 x 1-10nm

IDENTIFICATION - BIOCHEMICAL

Metabolic characteristics: substrates for growth

BERGEY’S MANUAL OF DETERMINATIVE BACTERIOLOGY

Bacteriologists BIBLE (Reference Text)

Divided into sections by TAXONOMY & CLASSIFICATION