Chapter 3 – Tools of the Laboratory
Methods of Culturing Microorganisms - The Five I’s – Fig 3.1
Five Basic Techniques to manipulate, grow, examine and characterize microorganisms
 Inoculation
 Incubation
 Isolation
 Inspection
 Identification
Inoculation: Producing a culture
 culture medium – a nutrient material prepared for the growth of microorganisms
 inoculation – the process of introducing a sample onto a culture medium
 inoculum – the microbes or sample that is placed onto a culture medium to initiate growth
 culture
o used as a verb – to grow microbes
o used as a noun – the observable growth; microbes that grow & multiply in a culture medium
 subculture – a culture that is made from a sample of a previous culture
 colony – a clone of bacterial cells growing on a solid culture medium; theoretically
arises from a single cell or clump of cells
Samples or specimens for culture – clinical samples ( blood, urine, CSF, other body fluids, sputum and
other respiratory samples, feces, skin sites, wounds, including surgical incisions) other samples – soil, water,
food, sewage, sinks, faucets, other inanimate objects
Isolation – Separating One Species from Another – Fig 3.2
The objective is to separate the bacteria in a sample into individual cells. Each cell will then grow into a small
clump called a colony – see definition above.
Requires: a culture medium, Petri dish, separation tools ( e.g. inoculating loops)
Obtaining Pure Cultures -- Isolation Techniques
A colony arises from a single cell or spore ( clones) – Fig 3.2
Streak Plate Method – Fig 3.3
 dilutes bacteria on the surface of an agar plate (Petri dish)
 most commonly used method
 use inoculating loop
o flame loop in between each quadrant that you make ; cool before streaking
o using the thin edge of the loop gives better isolation than the flat part
o hold the plate in the opposite hand while streaking ( not laying flat on the lab benchtop)
o keep plates closed between streaking; don’t talk, cough etc over the plates.
 colonies grow on the surface - usually get isolated colonies in 3rd or 4th quadrant
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Pour Plate and Spread Plate Method - See Fig. 3.3
if bacteria are present in large numbers – make a serial dilution
Serial dilutions – dilute by a series See Fig. 3.3
Pour Plate
o The dilution is made in tubes of melted agar
o Each tube is poured into a sterile, empty Petri dish, allowed to harden and incubated
o Count colonies ( choose plate w/ 25-250 colonies) – some colonies develop within the agar
o Multiply by dilution factor to get # cells / ml. of sample
o Advantage - Only counts viable cells
o Disadvantage – dilution errors; colonies within agar can’t be tested; heat sensitive
organisms
Spread Plate – The diluted, liquid sample is inoculated onto the surface of an agar plate and
spread evenly with a spreading tool – glass rod or plastic “hockey stick”
Types of Media
Physical States
 Liquid -- broths, milks, infusions ( e..g., nutrient broth, thioglycollate broth) –
 Solid - Agar (1-5%) is added to make the medium solid – Fig 3.5
o polysaccharide derived from seaweed ( red algae)
o most microbes cannot degrade agar
o liquefies at ~ 1000C ; solidifies ~ 400C ( will remain solid at incubator temp. of 35-370C)
o slants ; deeps ; Petri dishes (plates)
o firm surface – allow for colony formation and isolation ( Petri dishes)
 Semi-solid – Lower concentration of agar ( 0.3-0.5%) – Fig 3.4
o Clot-like, semi-solid consistency - Gelatin agar, motility agar
Chemical Content of Media
A. Ingredients for a growth medium
1. nutrients – C, N, S, P, peptone, proteins, amino acids, growth factors
2. moisture
3. adjusted pH ( buffers)
4. correct O2 level
5. sterility ( must be sterile)
B. Chemically Defined (Synthetic) Medium – See Table 3.2A
1. exact chemical composition is known
2. must contain organic growth factors for a carbon & energy source
3. not for “fastidious” organisms – these require many growth factors ( e.g. Neisseria spp.)
4. e.g., Euglena synthetic medium, fungal minimal medium
C. Complex (Nonsynthetic) Medium -- See Table 3.2B
1. exact chemical composition varies
2. used for most heterotrophic bacteria and fungi
3. energy, C, N, and S are supplied by protein
4. vitamins & growth factors are supplied in extracts from yeast, meats, plants, serum, etc.
5. e.g., nutrient broth or nutrient agar, blood agar, MacConkey
Functional Classification of Media
A. General purpose
1. usually complex (non-synthetic) ; gro broad spectrum of organisms
2. nutrient agar and broth, brain-heart infusion, trypticase soy agar
B. Enriched medium Fig 3-6
1. complex ( contain blood, serum, growth factors, etc.)
2. Good for fastidious organisms
3. Good for obtaining growth when bacterial numbers are low ; will increase numbers to a
detectable level
4. e.g. blood agar, chocolate agar
C. Selective Media – Fig 3-7, Table 3.3
1. suppress the growth of unwanted bacteria / allow growth of other bacteria
2. selective agents: pH, dyes, antibiotics, alcohol etc.
3. example: Mannitol Salt agar, or MacConkey – fig 3.9
D. Differential Media – – Fig 3-7, Table 3.4
1. can distinguish different types of bacteria
2. Media contain chemicals; different groups of bacteria react differently
3. Blood Agar – detects hemolysis of RBCs ( Fig. 3.6)
a) Alpha (partial hemolysis) ; Beta (complete hemolysis) ; gamma (no hemolysis)
4. MacConkey Agar – lactose fermenters (pink) from non-lactose fermenters ( clear)
5. Mannitol Salt Agar - mannitol fermenters (yellow) vs. non mannitol fermenters (red)
6. MacConkey & Mannitol salt are both selective and differential agars – Fig 3-8
E. Miscellaneous Media
1. Anaerobic Growth Media & Methods for Culturing Anaerobes
a) anaerobic organisms are killed by exposure to oxygen
b) Reducing media – contain ingredients that chemically combine with the oxygen
in the media ( remove it) – e.g., thioglycollate broth
2. Carbohydrate fermentation media – Fig 3.10
a) Contain sugars and a pH indicator
b) If sugars are fermented to acid, the pH indicator changes color
3. Transport Media
a) Maintain and preserve specimens that have to be held for a period fo time
before clinical analysis
b) Stuart’s or Amie’s transport media
F. Special Culture Techniques
1. Growth in live animals - e.g., Mycobacterium leprae / armadillos
2. Treponema pallidum – the syphilis spirochete ; cannot grow on artificial media
3. Rickettsia, chlamydiae – intracellular bacteria – must be grown in live cell culture
4. Viruses – must be grown in live cell culture
Incubation, Inspection and Identification
A. Incubation
 temperature-controlled environment ( usu 35°-37° C)
 control atmosphere
o with or without oxygen
o Capnophiles – require high concentrations of CO2
B. Inspection – Fig 3.11
 Pure culture – grows only a single known species of microorganism
 Subculture – to make a second-level culture from a well-isolated colony
 Mixed culture – has two or more identified or differentiated species of microorganisms
 Contaminated culture – has had a contaminant or unwanted organism introduced into it
C. Identification
 Colony appearance
 Microscopic examination
 Biochemical tests
 Genetic (DNA) analysis
 Immunological testing – test the isolate against known antibodies
 Animal inoculation
D. Maintenance and Disposal
 Cultures and specimens are biohazards ; Use proper disposal methods
o Steam sterilizing (autoclaving); incineration; hazardous waste disposal companies
 Maintenance – stock cultures are maintained for routine research, testing, and quality control
o Usually frozen or freeze-dried
o ATCC – American-Type Culture Collection