CULTURE MEDIA &
CULTURE METHODS
Prepared by: Ricci Alyssa S. Tumampil, SMT
HISTORY
* Bacteria have to be grown (cultured) for them to be identified.
* By appropriate procedures they have to be grown separately (isolated) on culture media and obtained as pure for study.
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* URINE / MEAT BROTH
* The original media used by LOUIS PASTEUR
* LIQUID MEDIUM
* Diffuse growth
* SOLID MEDIUM
* Discrete colonies
* COLONY
* Macroscopically visible collection of millions of bacteria originating from a single bacterial cell
* COOKED CUT POTATO
* By ROBERT KOCH
* Earliest solid medium
* GELATIN
* Not satisfactory
* Liquefy at 24 C
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AGAR
* FRAU HESSE
* Used for preparing solid medium
* Obtained from seaweeds
* No nutritive value
* Not affected by the growth of the bacteria
* Melts at 98 C
* Sets at 42 C
* 2% agar is employed in solid medium
TYPES OF CULTURE MEDIA
Based on…
1. CONSISTENCY
1. Solid Medium
2. Liquid Medium
3. Semi Solid Medium
2. CONSTITUENTS/INGREDIENTS
1. Simple Medium
2. Complex Medium
3. Synthetic or Defined Medium
3. SPECIAL MEDIA
1. Enriched Media
2. Enrichment Media
3. Selective Media
4. Indicator Media
5. Differential Media
6. Sugar Media
7. Transport Media
8. Media for Biochemical Reactions
1. Oxidase Test
2. Triple Sugar Iron Agar (TSI)
3. Indole Test
4. Citrate Utilization
5. Urease Test
4. OXYGEN REQUIREMENT
1. Aerobic Media
2. Anaerobic Media
1. Based on CONSISTENCY
* SOLID MEDIA
* Contains 2% agar
* Colony morphology, pigmentation, hemolysis can be appreciated
* Nutrient agar, Blood agar
* LIQUID MEDIA
* No agar
* For inoculum preparation, blood culture, for isolation of pathogens from a mixture
* Nutrient broth
* SEMI SOLID MEDIA
* Contains 0.5% agar
* Motility medium
2. Based on CONSTITUENTS/INGREDIENTS
* SIMPLE/BASAL MEDIA
* Nutrient broth
* Consist of:
* Peptone
* Meat Extract
* NaCl
* Nutrient agar
* Nutrient broth + 2% agar
* COMPLEX MEDIA
* Media other than basal media
* They have added ingredients
* Provide special nutrients
* SYNTHETIC/DEFINED MEDIA
* Media prepared from pure chemical substances and its exact composition is known
* Peptone Water
* 1% peptone + 0.5% NaCl in H2O
3. Based on SPECIAL MEDIA
* ENRICHED MEDIA
* Substances like blood, serum, egg are added to the basal medium
* Used to grow bacteria that are exacting in their nutritional needs
* Blood agar, Chocolate agar
* ENRICHMENT MEDIA
* Liquid media used to isolate pathogens from a mixed culture
* Media is incorporated with inhibitory substances to suppress the unwanted organisms
* Selenite F Broth
* Salmonella
* Shigella
* Alkaline Peptone Water
* Vibrio cholerae
* SELECTIVE MEDIA
* The inhibitory substance is added to a solid media
* Mac Conkey’s Medium
* Gram-negative bacteria
* Thiosulfate-Citrate Bile Salt Sucrose (TCBS) Agar
* Vibrio cholerae
* Lowestein-Jensen (LJ) Medium
* Mycobacterium tuberculosis
* Wilson and Blair Medium
* Salmonella typhi
* Potassium Tellurite Medium
* Corynebacterium diphtheriae
* INDICATOR MEDIA
* Contain an indicator which changes its color when a bacterium grows in them
* Blood agar
* Alpha-hemolysis
* Partial hemolysis
* Beta-hemolysis
* Complete hemolysis
* Gamma-hemolysis
* No hemolysis
* MacConkey’s medium
* Pink colonies = lactose fermenter
* Colorless colonies = non lactose fermenters
* Christensen’s urease medium
* Pink = positive
* Yellow = negative
* DIFFERENTIAL MEDIA
* Has substances incorporated in it enabling it to distinguish between bacteria.
* MacConkey’s medium - distinguish between lactose fermenters and non lactose fermenters
* Peptone
* Lactose
* Agar
* Neutral red
* Taurocholate
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* SUGAR MEDIA
* Media containing any fermentable substance.
* Glucose, arabinose, lactose, starch, etc.
* Media consists of 1% the sugar in peptone water
* Contain a small tube (Durham’s tube) for the detection of gas by the bacteria
* TRANSPORT MEDIA
* Media used for transporting the samples
* Delicate organisms may not survive the time taken for transporting the specimen without a transport media
* Stuart’s medium
* Non nutrient soft agar gel containing a reducing agent
* Buffered glycerol saline
* Enteric bacilli
* ANAEROBIC MEDIA
* Used to grow anaerobic organisms
* Robertson’s cooked meat medium
* Thioglycollate medium
* Most used in hospitals
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CULTURE MEDIA USED NEUTRAL RED
(SMaC)
* Salmonella Shigella Agar (SSA)
* MacConkey Agar
CULTURE MEDIA USED PHENOL RED
(MXCUT)
* Mannitol Salt Agar
* Xylose Lysine Deoxycholate
* Cystine Tryptic Agar (CTA)
* Urease
* Triple Sugar Iron (TSI)
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* MEDIA FOR BIOCHEMICAL REACTIONS
* They provide additional information for identification of the bacterium.
* The tests include:
* Oxidase Test
* Triple Sugar Iron Agar (TSI)
* Indole Test
* Citrate Utilization
* Urease Test
* OXIDASE TEST
* Detects the presence of an enzyme “oxidase” produced by certain bacteria which will reduce the dye tetramethyl-p-phenylenediamine dihydrochloride
* POSITIVE test = PURPLE color
* OXIDASE POSITIVE
* Pseudomonas
* Vibrio
* Neisseria
* OXIDASE NEGATIVE
* Salmonella
* Shigella
* TRIPLE SUGAR IRON (TSI) AGAR
* A composite media used to study different properties of bacterium
* Sugar fermentation
* Gas production
* H2S production
* Composed of:
* Peptone
* Yeast extract
* Agar
* Glucose
* Lactose
* Sucrose
* IRON SALT
* Ferric citrate indicates H2S production
* PHENOL RED is the indicator
* ORANGE RED medium with a slant and a butt
* pH of the medium is 7.4
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* TSI REACTIONS:
Yellow = Acid (A)
Pink = Alkaline (K)
* Yellow Slant/Yellow Butt (A/A) = Lactose fermenters
* Pink Slant/Yellow Butt (K/A) = Non lactose fermenters
* Pink Slant/No colour change (K/K) = Non fermenters
* Black Colour = H2S Production
* Gas bubbles or crack in the medium = Gas production
* RAPID LACTOSE FERMENTERS
(A/AG H2S-)
* Escherichia coli
* Klebsiella spp.
* Enterobacter spp.
* NON LACTOSE FERMENTERS
(K/AG H2S+)
* Salmonella spp.
* Proteus spp.
* Arizonae spp.
* Citrobacter spp.
* Edwardsiella spp.
* NON LACTOSE/SUCROSE FERMENTERS H2S NON-PRODUCER
(K/A H2S-)
* Shigella spp.
* NON-FERMENTER/OXIDIZER
(K/K H2S-)
* Non-fermentative bacteria:
Pseudomonas spp.
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* INDOLE TEST
* Used to detect indole production by the organism
* They produce indole from tryptophan present in peptone water
* After overnight, a few drops of indole reagent (Kovac’s reagent) is added
* Indole POSITIVE test = PINK RING
* Morganella spp.
* Proteus vulgaris
* Escherichia coli
* Klebsiella oxytocin
* Providencia spp.
* Edwardsiella tarda
* Citrobacter koseri
* Plesiomonas spp.
* Indole NEGATIVE test = YELLOW RING
* CITRATE UTILIZATION
* Done in Simmon’s Citrate medium
* Detect the ability of certain bacteria to utilize citrate as the sole source of carbon
* Contains sodium citrate and bromothymol blue as the indicator
* If CITRATE is UTILIZED, ALKALI is produced which turns the medium to blue
* Citrate POSITIVE = BLUE colour
* Klebsiella spp.
* Citrate NEGATIVE = GREEN colour
* Escherichia coli
* UREASE TEST
* Done in Christensen’s urease medium
* Detect organisms that produce urease
* Urease produced by organisms split urea into ammonia and CO2
* Urease POSITIVE = PINK colour
* Urease NEGATIVE = YELLOW colour
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RAPID UREASE
(TPUNCH)
* Trichophyton mentagrophytes
* Proteus, Providencia, Morganella (PPM)
* Ureaplasma
* Nocardia
* Cryptococcus neoformans
* Helicobacter pylori
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SLOW UREASE
(CKEYS)
* Citrobacter
* Klebsiella
* Enterobacter gergoviae
* Yersinia enterocolitica
* Serratia
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CULTURE METHODS
* Employed depend on the purpose for which they are intended.
* Indications for culture are:
* To isolate bacteria in pure cultures
* To demonstrate their properties
* To obtain sufficient growth for the preparation of antigens and for other tests.
* For bacteriophage and bacteriocin susceptibility
* To determine sensitivity to antibiotics
* To estimate viable counts
* Maintain stock cultures
* Includes:
* Streak culture
* Lawn culture
* Stroke culture
* Stab culture
* Pour plate method
* Liquid culture
* Anaerobic culture methods
* STREAK CULTURE
* Used for isolation of bacteria in pure culture from clinical specimens
* Uses platinum wire or nichrome wire
* One loopful of specimen is transferred onto the surface of a well dried plate
* Spread over a small area at the periphery
* The inoculum then distributed thinly over the plate with a loop in a series of parallel lines on different segments of the plate
* On incubation, separated colonies are obtained over the last series of streaks
* LAWN CULTURE
* Provides a uniform surface growth of the bacterium
* Uses:
* For bacteriophage typing
* Antibiotic sensitivity testing
* In the preparation of bacterial antigens and vaccines
* Prepared by flooding the surface of the plate with a liquid suspension of the bacterium
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* STROKE CULTURE
* Is made in tubes containing agar slope/slant
* For slide agglutination and other diagnostic tests
* STAB CULTURE
* Prepared by puncturing a suitable medium
* Gelatin or glucose agar with long, straight, charged wire
* Uses:
* Demonstration of gelatin liquefaction
* Oxygen requirements of the bacterium under study
* Maintenance of stroke culture
* POUR PLATE METHOD
* Agar is melted (15 mL) and cooled to
45 C
* 1 mL of the inoculum is added to the molten agar
* Mix well and pour to a sterile petri dish
* Allow it to set
* Incubate at 37 C, colonies will be distributed throughout the depth of the medium
* Uses:
* Gives an estimate of the bible bacterial count in a suspension
* For the quantitative urine cultures
* LIQUID CULTURE
* Are inoculated by touching with a charged loop or by adding the inoculum with pipettes or syringes
* Uses:
* Blood culture
* Sterility test
* Continuous culture methods
* Disadvantages:
* It does not provide a pure culture from mixed inocula
* ANAEROBIC CULTURE METHODS
* Anaerobic bacteria differ in their requirement and sensitivity to oxygen
* Clostridium tetani is a strict anaerobe– grows at an oxygen tension <2 mmHg
* METHODS:
* Production of vacuum
* Incubate the cultures in a vacuum desiccator
* Displacement of oxygen with other gases
* Displacement of oxygen with hydrogen, nitrogen, helium, or CO2
* Candle jar
* Chemical method
* Alkaline pyrogallol absorbs oxygen
* Biological method
* Reduction of medium
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McIntosh - Fildes’ Anaerobic Jar
* Consists of a metal jar or glass jar with a metal lid which can be clamped air tight
* The lid has 2 tubes: Gas inlet and Gas outlet
* The lid has two terminals connected to electrical supply
* Under the lid is a small grooved porcelain spool, wrapped with a layer of palladinised asbestos
* WORKING
* Inoculated plates are placed inside the jar and the lid clamped air tight
* The outlet tube is connected to a vacuum pump and the air inside is evacuated
* The outlet tap is then closed and the inlet tube is connected to a hydrogen supply
* After the jar is filled with hydrogen, the electric terminals are connected to a current supply, so that the pallasidinised asbestos is heated
* Act as a catalyst for the combination of hydrogen with residual oxygen
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Gaspak
* Commercially available disposable envelope
* Contain chemicals which generate H2 and CO2 on addition of water
* Cold catalyst - in the envelope
* Indicator - reduced methylene blue
* Colourless = anaerobic
* Blue colour = on exposure to oxygen
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