Culture Media & Culture Methods PDF

CULTURE MEDIA & CULTURE METHODS § 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. History § The original media used by Louis Pasteur – urine or meat broth...

CULTURE MEDIA & CULTURE METHODS § 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. History § The original media used by Louis Pasteur – urine or meat broth § Liquid medium – diffuse growth § Solid medium – discrete colonies. CULTURE MEDIA — artificially prepared media which contains all essential nutrients to provide for growth and multiplication of particular microorganism.  CULTURE MEDIA - are nutrient solutions or solid preparations used to grow, support, and sustain microorganisms such as bacteria, fungi, or cells in laboratory settings - provide essential nutrients like carbohydrates, proteins, vitamins, minerals, and growth factors necessary for microbial growth - Culture media can be categorized based on their composition, use, and properties into general- purpose, selective, differential, or enriched media, depending on the specific needs of the microorganisms being cultured Common Types of CULTURE MEDIA General-purpose media: Support the growth of a wide range of microorganisms (e.g., Nutrient Agar). Selective media: Promote the growth of certain microorganisms while inhibiting others (e.g., MacConkey Agar). Differential media: Distinguish between organisms based on metabolic activity (e.g., Blood Agar). Enriched media: Contain additional nutrients to support the growth of fastidious organisms (e.g., Chocolate Agar).  Culture Medium Category Uses/Indications Culture Medium Category Uses/Indications Used for the growth Buffered Charcoal Yeast Enriched Used for the cultivation of non- of Legionella species and Extract (BCYE) fastidious bacteria. Supports the other fastidious organisms. Nutrient Agar (NA) General-purpose growth of a wide variety of Used to cultivate anaerobic microorganisms. and microaerophilic Thioglycollate Broth Enriched Used for the growth of fastidious organisms; supports the organisms and to detect hemolytic growth of obligate anaerobes. Blood Agar (BA) Enriched, Differential activity (alpha, beta, or gamma Used for the growth of a hemolysis). wide variety of non-fastidious Tryptic Soy Agar (TSA) General-purpose Selective for Gram-negative bacteria organisms; commonly used and differentiates between lactose for general bacterial culture. MacConkey Agar (MAC) Selective, Differential fermenters (pink colonies) and non- Selective lactose fermenters (colorless for Mycobacterium species, colonies). Lowenstein-Jensen (LJ) Selective particularly M. tuberculosis; Medium Selective for Gram-negative bacteria contains inhibitors for other Eosin Methylene Blue (EMB) and differentiates lactose fermenters bacteria. Selective, Differential Agar (dark colonies, often with metallic sheen) from non-fermenters. Used to enrich Salmonella species Selenite F Broth Enrichment Supports the growth of fastidious from stool samples before Chocolate Agar (CA) Enriched organisms, such as Haemophilus plating on selective media. influenzae and Neisseria species. Selective for Campylobacter species; Selective for Staphylococcus species CAMPY-BAP Agar Selective contains antibiotics to inhibit (high salt concentration) and Mannitol Salt Agar (MSA) Selective, Differential differentiates S. aureus (mannitol other intestinal flora. fermenter, yellow colonies) from Selective for Enterococci and other staphylococci (red colonies). Group D Streptococci; Bile Esculin Agar (BEA) Selective, Differential hydrolysis of esculin turns Used for the selective isolation the medium black. Thayer-Martin Agar Enriched, Selective of Neisseria gonorrhoeae and Neisseria meningitidis. Used for the isolation of Corynebacterium Selective for fungi (yeasts and diphtheriae; differentiates Sabouraud Dextrose Agar molds); inhibits bacterial growth due Tinsdale Agar Selective, Differential Selective based on the production of (SDA) to low pH and high glucose concentration. black colonies due to tellurite reduction. Selective for Gram-negative enteric Differentiates bacteria based Xylose Lysine Deoxycholate pathogens and Selective, Differential on their ability to ferment (XLD) Agar differentiates Salmonella (black Triple Sugar Iron (TSI) colonies) and Shigella (red colonies). Differential glucose, lactose, or sucrose Agar and produce hydrogen sulfide Selective for Pseudomonas aeruginosa; (H₂S). Cetrimide Agar Selective enhances pigment production in Pseudomonas species. Used to isolate and differentiate Salmonella (black Hektoen Enteric Agar (HEA) Selective, Differential colonies) and Shigella (green colonies) from other enteric organisms. General Characteristics of culture Media ØMust give satisfactory and rapid growth from single culture ØShould maintain pH during storage ØReasonably cheap and easily available ØMaintain sterility through expt.  USES OF MEDIA üCultivation and isolation üIdentification üSterility test üPreservative efficacy test üEvaluation of disinfectant üTo check the antimicrobial susceptibility 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 24oC Agar — Fannie Hesse — Used for preparing solid medium — Obtained from seaweeds. — No nutritive value — Not affected by the growth of the bacteria. — Melts at 98oC & sets at 42oC — 2% agar is employed in solid medium  Types of culture media I. Based on their consistency a) solid medium b) liquid medium c) semi solid medium II. Based on the constituents/ ingredients a) simple medium b) complex medium c) synthetic or defined medium d) Special media Special media ◦ Enriched media ◦ Enrichment media ◦ Selective media ◦ Indicator media ◦ Differential media ◦ Sugar media ◦ Transport media ◦ Media for biochemical reactions III.Based on Oxygen requirement - Aerobic media - Anaerobic media Solid media – contains 2% agar § Colony morphology, pigmentation, hemolysis can be appreciated. § Eg: Nutrient agar, Blood agar Liquid media – no agar. § For inoculum preparation, Blood culture, for the isolation of pathogens from a mixture. § Eg: Nutrient broth Semi solid medium – 0.5% agar. § Eg: Motility medium Microbiological Media — Liquid (broth) vs. semisolid media ◦ Liquid medium – Components are dissolved in water and sterilized ◦ Semisolid medium – A medium to which has been added a gelling agent – Agar (most commonly used) – Gelatin – Silica gel (used when a non-organic gelling agent is required) Solid medium Semi solid media Liquid media Simple media / basal media - Eg: Nutrient broth, Nutrient agar - NB consists of peptone, meat extract, NaCl, - NB + 2% agar = Nutrient agar  Simple media / basal media — Substances such as blood, egg, serum are added to the basal medium — Used to grow bacteria which are more exacting in their nutritional needs — Example: Blood agar – hemolytic properties Chocolate agar – to supply blood factors like X factor (Hemin) and V factor (NAD) Complex media — Media other than basal media. — They have added ingredients. — Exact chemical composition of ingredients of culture medium is not known — Widely used for the cultivation of bacteria for diagnostic purposes — Provide special nutrients — highly nutritious substances. Synthetic or defined media — Media prepared from pure chemical substances and its exact composition is known — precise amounts of highly purified chemicals — Eg: peptone water – 1% peptone + 0.5% NaCl in water  Synthetic or defined media — Prepared from pure chemical substances and the exact composition of the medium is known — Used for various special studies such as metabolic requirements — Example: Dubo’s medium Enriched media § Substances like blood, serum, egg are added to the basal medium. § Used to grow bacteria that are exacting in their nutritional needs. § Eg: Blood agar, Chocolate agar Blood agar Chocolate agar Enrichment media § Liquid media used to isolate pathogens from a mixed culture. § Is used to encourage the growth of a particular microorganism in a mixed culture § Media is incorporated with inhibitory substances to suppress the unwanted organism. § Eg: ◦ Selenite F Broth – for the isolation of Salmonella, Shigella ◦ Alkaline Peptone Water – for Vibrio cholerae Selective media — The inhibitory substance is added to a solid media. — Contains one or more agents that inhibit the growth of a certain microbe and thereby encourages, or selects, a specific microbe E.g.: — Mannitol Salt Agar [MSA] encourages the growth of Staphylococcus aureus. MSA contain 7.5% NaCl which inhibit the growth of other Gram +ve bacteria — MacConkey’s medium for gram negative bacteria — TCBS – for Vibrio cholerae — Lowenstein Jensen medium – Mycobacterium tuberculosis — Wilson and Blair medium – Salmonella Typhi — Potassium tellurite medium – Diphtheria bacilli Potassium Tellurite media LJ media Mac Conkey’s medium TCBS Indicator media — These media contain an indicator which changes its color when a bacterium grows in them. — E.g.: ◦ Blood agar ◦ MacConkey’s medium ◦ Christensen’s urease medium Differential media § A media which has substances incorporated in it enabling it to distinguish between bacteria. § E.g.: MacConkey’s medium ◦ Peptone ◦ Lactose ◦ Agar ◦ Neutral red ◦ Taurocholate § Distinguish between lactose fermenters & non lactose fermenters.  § Lactose fermenters – Pink colonies § Non lactose fermenters – colourless colonies Mac Conkey’s medium Sugar media § Media containing any fermentable substance. § Eg: glucose, arabinose, lactose, starch etc. § Media consists of 1% of 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. § Eg: ◦ Stuart’s medium – non nutrient soft agar gel containing a reducing agent ◦ Buffered glycerol saline – enteric bacilli  CULTURE METHODS — Culture methods employed depend on the purpose for which they are intended. — The 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 & bacteriocin susceptibility. ◦ To determine sensitivity to antibiotics. ◦ To estimate viable counts. ◦ Maintain stock cultures. Culture methods include: § Streak culture § Lawn culture § Stroke culture § Stab culture § Pour plate method § Liquid culture § Anaerobic culture methods STREAK CULTURE — Used for the isolation of bacteria in pure culture from clinical specimens. — Platinum wire or Nichrome wire is used. — One loopful of the specimen is transferred onto the surface of a well dried plate. — Spread over a small area at the periphery. — The inoculum is then distributed thinly over the plate by streaking it with a loop in a series of parallel lines in different segments of the plate. — On incubation, separated colonies are obtained over the last series of streaks. A petri plate with bacterial growth showing the distinction between isolated colonies and non-isolated colonies. A three sector T streak of Serratia marcescens grown on trypticase soy agar. This illustrates a streak plate which has many isolated colonies. This plate illustrates a streak plate which did not achieve isolation, and which would not be considered a good streak plate example 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. — Lawn cultures are prepared by flooding the surface of the plate with a liquid suspension of the bacterium. Lawn Culture - Antibiotic sensitivity testing STROKE CULTURE — Stroke culture is made in tubes containing agar slope / slant. — Uses ◦ Provide a pure growth of bacterium for slide agglutination and other diagnostic tests. STAB CULTURE — Prepared by puncturing a suitable medium – gelatin or glucose agar with a long, straight, charged wire. — Uses ◦ Demonstration of gelatin liquefaction. ◦ Oxygen requirements of the bacterium under study. ◦ Maintenance of stock cultures. Gelatin liquefaction Oxidation – Fermentation medium POUR PLATE CULTURE — Agar medium is melted (15 ml) and cooled to 45oC. — 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 37oC, colonies will be distributed throughout the depth of the medium. — Uses ◦ Gives an estimate of the viable bacterial count in a suspension. ◦ For the quantitative urine cultures. LIQUID CULTURES — Liquid cultures are inoculated by touching with a charged loop or by adding the inoculum with pipettes or syringes. — Uses ◦ Blood culture ◦ Sterility tests ◦ Continuous culture methods — Disadvantage ◦ It does not provide a pure culture from mixed inocula. Blood culture bottles