3. 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
5. PHYSICAL CHEMICAL FUNCTIONAL
STATE COMPOSITION TYPE (Purpose)
Liquid Synthetic General Purpose
(Chemically)
Semi-solid Enriched
Solid (Liquid) Selective
Solid Non-synthetic Differential
(not chemical)
Anaerobic
Growth
Specimen
transport
Assay
Enumeration
6. 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
7. SEMI SOLID MEDIA
clot like consistency, contain solidifying agent
(agar/gelatin - 0.3-0.5%)
Used to determine motility, localize reaction at specific
sites
8. 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
9.
10. 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
11. GENERAL PURPOSE MEDIA
Used for a broad spectrum of microbes, non synthetic
Examples: Brain-heart infusion
Tryptose soy agar
Tryptose soy broth
12. ENRICHMENT MEDIUM
complex organic substances : blood, serum, growth factors
Used for FASTIDIOUS ORGANISMS
Streptococcus pneumoniae
Requires blood - sterile horse, sheep or rabbit
13. 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
14. 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
15.
16.
17.
18. 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
19. EXAMPLES
MAcCONKEY AGAR - lactose + neutral red
E. coli produces acid, metabolizes lactose
RED-PINK colonies
Salmonella sp produce no acid
OFF WHITE colonies
22. 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
24. 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
25. 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
26. 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
27. INCUBATION
Chamber (INCUBATOR)
temperature & atmospheric gas controlled
LAB INCUBATORS : 20 - 40oC
Aerobic or Anaerobic
INCUBATION PERIOD : hours-several weeks depending
upon the organism
28. 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)
30. MICROSCOPICALLY
individual cells within a colony
Evidence of cellular morphology: size, shape, details of
structure
allows for IDENTIFICATION
31. AIMS
to provide adequate MAGNIFICATION, RESOLUTION and
CLARITY of IMAGE
32. TOTAL POWER OF MAGNIFICATION
Power of Power of Total
Objective Ocular Magnification
40x high (dry) 10x 400x
100x oil imm 10x 1000x
10x low 20x 200x
power
33.
34. SUB-CULTURE common microbiological procedure
allows for a pure STOCK-CULTURE of organism
DISPOSAL OF CULTURES
most important - if presents a biological hazard
Autoclaving - steam sterilization
Incineration - burning
Radiation - X or rays
Disinfection - chemical
35. 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
36. 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
37. FIXED PREPARATIONS
Advantage: Permanent mount, long term study
Smear technique : Developed by Koch >100yrs ago
Disadvantage: KILLS specimen
38. 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”
40. POSITIVE STAINING
+ve stain - sticks to specimen providing colour
Bacillus cereus stained with carbol fuschin (1300x)
41. NEGATIVE STAINING
-ve stain - (reverse) settles around specimen
boundary forms a silhouette (stains the glass slide)
Escherichia coli stained with India ink (1300x)
42. 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
43. 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
46. ACID FAST STAIN - Paul Ehrlich
similar to Gram’s, used with resistant bacteria
Acid-fast Bacteria : Pink
Non Acid-fast bacteria : Blue
Mycobacterium : tuberculosis
50. ENDOSPORE STAIN
similar to Acid-fast
Distinguishes between bacteria producing spores and
those that do not
For Identification of Bacillus sp., Clostridium sp.
54. 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
59. 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
61. 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
72. 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
