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Microbial growth

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					L7: Culture and Identification
Prepared by Dr. Marianne Watts

objectives
Understand how the characteristics of microbial metabolism may be used in their identification Know composition/design of several different types selective and differential media Understand how these media work

Why culture microbes ?

Things to consider: general

Lab Culture: batch Liquid or solid media ?
Liquid media rapid growth large volume contamination Solid media isolation from mixed cultures visualization and identification Solidifying agent: Agar advantages
melts at 100 degrees but remains liquid until cooled to 40 degrees Relatively inert Transparent

Solid medium: types

Culture Media
Complex v Chemically Defined
Complex

contain materials of biological origin Egs…………………………..
Used to cultivate organisms that require a lot of growth factors
Grow most heterotrophic organisms

Chemically Defined Composed of pure biochemicals Growth of specific
heterotrophs chemoautotrophs, photoautotrophs

Culture of pathogens
adapted for growth in animal tissues What kind of media would you use? For bowel organisms? Skin organisms? ‘fastidious’ pathogens?
Eg Neisseria Haemophilus

Some are ‘unculturable’

So….. how to separate?
Selective
Selective agents?

Differential
Differential agents?

enriched Enrichment reducing

Isolation scheme
Source material Enrichment (broth) Plating for isolation
Selective/differential/enriched/etc

Characterisation and ID Antibiotic sensitivity

If really low nos suspected
Resuscitation (broth) Pre-enrichment (broth)

Culture media: examples

Complex or chem defined? What org would you use these for?

Chemically Defined media examples: which is for what type of org?

Enriched medium: added growth factors

What does chocolatising the blood achieve?

Blood agar can be differential

beta

apha

Different types of haemolysis

Type of haemolyis?

Cl. perfringens

Selective Mackonkey agar : contains bile salts

what type of organisms are growing?

Mac contains: lactose, ph indicator dye : differential

Mac: LF or NLF? ? Pure culture

Klebsiella sp

MSA: selective & differential for for Staphylococci: why?

Contains salt, Mannitol, pH indicator dye

Selective agent? differential agent? S. epidermidis or S. aureus: Which is growing where?

TCBS : Thiosulphate, citrate, bile salts, sucrose: pH 8.6
Selective agent Diff agent?

Do not autoclave

V. cholerae ferments sucrose

XLD: Xylose, lysine deoxycholate

Selective & differential agents?

Identification

Identification: overview
microscopic observations, Such as…….. Culture media Selective & differential reflect environment of organism Oxygen requirements (reflects metabolism) biochemical tests

distinguish species among genera (1st & 2nd stage tests C& S).
immunological tests

distinguishes strains within a single species

Brief clinical micro ID scheme
Specimens come in: GP, clinics or hospital Swabs: Throat, Wound, Vaginal, urethral Faeces Urine spit Blood CSF Clinical ID: needs to be rapid Do Gram (always), ZN (if sputum) Make a decision on which media to use Several selective and non-selective Incubate (O2 ANO2 etc) Next day: ID and do spot tests to confirm from pure culture on non-selective agar Further tests if necessary to confirm

Gram +ve: morphology important

Gram -ve

ZN stain: AAFB

CytoJournal 2004, 1:6 doi:10.1186/1742-6413-1-6

Pure culture technique: why do we need pure culture?
Colony isolation
Streaking pouring spreading

subculture
restreak an isolated colony to make sure that an isolated colony is truly derived from only a single cell Then characterize

inoculate into differential medium
what type of nutrients they require or can use, what types of by-products they produce Antibiotic sensitivity

Differentiation: morphology, margin, texture, pigmantation

Pigmentation v fermentation

Micrococcus and Serratia sp

Rough & smooth:Pseudomonas luteola
Citation: Forest W. Arnold, Carmen V. Sciortino, Keller A. Riede: New Associations With Pseudomonas Luteola Bacteremia: A Veteran With A History Of Tick Bites And A Trauma Patient With Pneumonia. The Internet Journal of Infectious Diseases. 2005. Volume 4 Number 2.

24 h culture

48 h culture

ID based on oxygen relationship
O-F test Thioglycolate medium Sugar fermentation

OF medium

ID on Aerobic or anaerobic growth
strict (or obligate) aerobes strict (or obligate) anaerobes facultative anaerobes aerotolerant anaerobe Microaerophile

Thioglycollate Medium: glucose & reducing agent
Which tube has which O2 relationship?
STRICT(OBLIGATE)ANAEROBE STRICT(OBLIGATE)AEROBE FACULTATIVE ANAEROBE AERO TOLERANT ANAEROBE

Other sugars? Fermentation media
a sugar Peptone Only a little. Why? pH indicator: Bromcresol purple, phenol red turn yellow under acidic conditions

Fermentation results

Don’t forget other end products are possible

Other basic ID tests based on enzymes

catalase

staphs

streps

Coagulase Test
coagulase test used to distinguish between Staphylococcus aureus and non-pathogenic staphylococci. Slide: bound coagulase Tube: free coagulase See methods book

Oxidase test
Tests for Cytochrome c oxidase This can oxidize the reagent Methylphenylenediamine

Positive oxidase reaction = purple color. Oxidase negative organisms cause little or no color change.

Motility medium: various types

questions
Pre-labs Tutorials Prac reviews prac 2 through to prac 8


				
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