MICROBIOLOGICAL METHODS
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