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