This document discusses culture media and culture methods used for growing bacteria in the laboratory. It describes the basic constituents of culture media including water, electrolytes, peptone, agar, meat extract, yeast extract, and blood/serum. Agar is preferred over gelatin as a solidifying agent. Common types of media are discussed such as basal media, enriched media, enrichment broth, selective media, differential media, transport media, and anaerobic media. Methods for streaking, lawn/carpet, stroke, and stab cultures as well as liquid culture techniques are also outlined.

1. CULTURE MEDIA
AND CULTURE
METHODS

2. CULTURE MEDIA
 BASIC CONSTITUENTS:
1. Water – distilled/portable water, low mineral content
2. Electrolytes
3. Peptone
• Source - lean meat or heart muscle or soya flour digested by pepsin
• Constituents – proteoses, amino acids, vitamins(growth factors)
4. Agar – solidifying agent
• Source – Seaweeds
• Components – Cell wall derived polysaccharides, some proteins, fatty acids, calcium and magnesium
• Concentration – 1-2% (solid agar), 0.5% (semisolid agar), 6% (inhibit Proteus swarming)
• Preparation – agar added to water, boiled (95 degrees), mixture dissolved, sterilized, cooled to 45
degrees (melts), poured into petridish, allowed to set for 20 min

3. 5. Meat extract
6. Yeast extract
7. Malt extract – maltose, starch, dextrose etc
8. Blood and serum – components of enriched media, extra nutrition to fastidious organisms
• 5- 10 % sheep blood
• Aseptic collection, rendered noncoagulable
• Serum sterilized by filtration after collection
 WHY AGAR AND NOT GELATIN?
• Agar – bacteriologically inert
• Gelatin – liquified by many bacteria
• Agar – melts at 98 degrees, Gelatin – melts at 24 degrees (liquid at room temperature)

4.  ROUTINE LAB MEDIA :
1. Simple/basal media
2. Enriched media
3. Enrichment broth
4. Selective media
5. Differential media
6. Transport media
7. Anaerobic media

5. 1. SIMPLE/BASAL MEDIA
• Testing non fastidious bacteria
• Base for preparation of many other media
• For studying bacterial growth curve
• Preferred for biochemical tests, studying colony morphology etc
1. Peptone water: Peptone (I%) + NaCl (0.5%) + water
2. Nutrient broth: peptone water+ meat extract (I%). lt is available in three forms: (1)
meat extract, (2) meat infusion, (3) meat digest broth
3. Nutrient agar: nutrient broth+ 2% agar
4. Semisolid medium: concentration of agar reduced to 0.2 to 0.5 %

8. 2. ENRICHED MEDIA
• Basal medium + additional nutrients (egg/ serum/ blood )
1. Blood agar : 5-10% of sheep blood to the molten nutrient agar at 45’C, tests
hemolytic property of bacteria
2. Chocolate agar : heated blood agar; 5- 10% of sheep blood to the molten nutrient
agar at 70’ C, RBCs lysed, brown colour, more nutritious than blood agar (eg : used
for Haemophilus influenzae)
3. Loeffler’s serum slope : contains serum, used for Corynebacterium diphteriae
4. Blood culture media
• Monophasic medium – Brain heart infusion broth (BHI)
• Biphasic medium – liquid phase (BHI broth), solid phase (BHI agar)

12. 3. ENRICHMENT BROTH
• Liquid broth + inhibiting agents
1. Tetrathionate broth – Salmonella Typhi
2. Gram-negative broth - isolation of Shigella
3. Selenite F broth- isolation of Shigella
4. Alkaline peptone water (APW)- Vibrio cholerae

15. 4. SELECTIVE MEDIA
• Solid media + inhibiting substances
1. Lowenstein-Jensen (LJ) medium - Mycobacterium tuberculosis
2. Thiosulphate citrate bile sail sucrose (TCBS) agar - Vibrio species
3. DCA (deoxycholate citrate agar) - enteric pathogens, Salmonella and Shigella from
stool
4. XLD (Xylose lysine deoxycholate) agar - same as DCA
5. Potassium tellurite agar (PTA) - Corynebacterium diphtheriae
6. Wilson Blair bismuth sulfite medium: It is used for isolation of Salmonella Typhi.

19. 5. DIFFERENTIAL MEDIA
• To differentiate between two groups using an indicator
1. MacConkey agar – for enteric gram negative organisms
• Differentiates lactose fermenters (LF eg; E coli) from non lactose fermenters (NLF eg;
shigella)
2. CLED agar - same as MacConkey but less inhibitory, allows gram positive, can
prevent swarming of proteus

21. 6. TRANSPORT MEDIA
• To preserve delicate organisms or if any delay; no division, only remain viable
1. Streptococcus - Pike's medium
2. Neisseria - Amies medium and Stuart's medium
3. Vibrio cholerae - • VR (Venkatraman-Ramakrishnan) medium
• Autoclaved sea water
• Cary Blair medium
4. Shigella, Salmonella- • Buffered glycerol saline
• Cary Blair medium

23. 7. ANAEROBIC MEDIA
• reducing substances which take up O2, for obligate anaerobes eg: Clostridium
1. Robertson's cooked meat (RCM) broth - chopped meat particles (beef heart), which provide
glutathione (reducing substance)
2. Thioglycollate broth
3. Anaerobic blood agar BHIS agar (Brain-heart infusion agar) with supplements ( vitamin K and
hemin)
4. Neomycin blood agar
5. Egg yolk agar
6. Phenyl ethyl agar
7. Bacteroides bile esculin agar (BBE agar)

25. CULTURE METHODS
 LOOPS AND STRAIGHT WIRES:
• BACTERIOLOGICAL LOOPS –
o Internal diameter of 2-4 mm
o Streaking culture plates
• BACTERIOLOGICAL STRAIGHT WIRES – for stroke and stab cultures
STERILE SWABS – for lawn culture
 inoculating straight wire or loop is first heated in the Bunsen flame by making it red hot and then
made cool waiting for 10 seconds.
 procedure to be carried out in biological safety cabinet

28.  STREAK CULTURE

29.  LAWN/ CARPET CULTURE
• METHODS :
1. SWABBING
2. FLOODING
• USES :
1. Antimicrobial susceptibility testing by disk diffusion method
2. Bacteriophage typing
3. Large bacterial growth for preparation of antigens and vaccines

31.  STROKE CULTURE
• On agar slopes, for diagnostic tests
• Eg : for citrate test and urease test
 STAB CULTURE
• On semisolid medium with a straight wire
• USES :
1. Oxidation fermentation test, TSI test, motility testing
2. Maintaining stock cultures

32.  LIQUID CULTURE
• BACTERIAL GROWTH – detected by turbidity
1. Uniform
2. Granular at the bottom
3. Surface pellicles
• USES :
1. Blood culture
2. Sterility testing
3. Water analysis