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Culture is the term for microorganisms grown in the lab. A culture medium provides nutrients to support microbial growth. Specialized media are used for different purposes like enrichment, selection, or differentiation. Diagnostic cultures identify pathogens from samples and common tests include urine, stool, genital and skin samples. Culture techniques allow isolation and study of microbes and are essential tools in medical microbiology.
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- 1. CULTURE TECHNIQUES – DIAGNOSTIC TOOLS IN MEDICAL MICROBIOLOGY Dr. Shikha Thakur Assistant Professor, Department of Biotechnology, Thakur College of Science and Commerce, Mumbai
- 2. CULTURE AND THE MEDIUM Culture : It is the term given to microorganisms that are cultivated in the lab for the purpose of identifying and studying them. Cultivation : The propagation of living organisms, applied especially to the growth of microorganisms or other cells in artificial media. Medium : Is the term given to the combination of ingredients that will support the growth and cultivation of microorganisms by providing all the essential nutrients required for the growth (i.e. multiplication) in order to cultivate these microorganisms in large number to study them.
- 3. Microbiological culture medium : which are used for growing microorganisms, such as bacteria or yeast. The most common growth media for microorganisms are nutrient broths and agar plates Specialized media are sometimes required for microorganism and cell culture growth. CULTURE MEDIA A culture medium is a solid or liquid preparation used to grow, transport, and store microorganisms.
- 4. Can be used Enrich the numbers of bacteria. Select for certain bacteria and suppress others. Differentiate among different kinds of bacteria. Isolation, identification and long-term storage of pure cultures BASIC REQUIREMENT OF CULTURE MEDIA When culturing bacteria, it is very important to provide similar environmental and nutritional conditions that exist in its natural habitat.
- 5. CHEMICAL REQUIREMENTS (NUTRITIONAL FACTORS) Nutrients- water, source of Carbon and energy, source of Nitrogen Mineral salts –Sulphate, phosphates, chlorides & carbonates of K, Mg & Ca. Organic growth factor like factors- ryptophan for Salmonella typhi- X & V factors for H. influenzae Vitamins (e.g. folic acid, vitamin B-12, vitamin K) Oxygen PHYSICAL REQUIREMENTS A suitable pH Temperature Hydrostatic Pressure Osmotic pressure
- 6. Preparation and storage of culture media Wash hands & wear gloves • Sterilize all equipments • Pour D/W in glassware (required amount) • Add powder ingredients (required amount) • Heat to dissolve completely • Autoclave • Dispense the medium into tubes ,bottles &plates • Store at required temperature • Sterilizing Culture Media
- 7. NEED FOR CULTURE MEDIA It is usually essential to obtain a culture by growing the organism in an artificial medium. If more than one species or type of organism are present each requires to be carefully separated or isolated on culture media and obtained as pure culture for study . Several organism need the determination of Antibiotic sensitivity pattern for optimal antibiotic selection Medium → Nutrients → support growth TYPES OF INFECTIONS THAT CAN BE DIAGNOSED Diagnostic cultures are commonly used to identify infectious microbes from samples isolated from urine ( urinary tract infections), stool (diarrheal and food-borne diseases), genital tract ( STD), throat ( strep throat), and skin (skin infections).
- 8. HISTORY The original media used by Louis Pasteur – urine or meat broth 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(1881) – earliest solid medium Gelatin – not satisfactory - liquefy at 24oC
- 9. Agar(1882) Frau Hesse Used for preparing solid medium It is suphated polymercomposedof mainly D-galactose, anhydro-L galactose and D-glucuronic acid Obtained from seaweeds ( red algae)- Gelidium. No nutritive value Not affected by the growth of the bacteria. Melts at 96oC & sets at 42oC 2% agar is employed in solid medium
- 10. CLASSIFICATION BASED ON PHYSICAL STATE( based on their consistency) BASED ON PRESENCE OF MOLECULAR OXYGEN AND REDUCING SUBSTANCES BASED ON NUTRITIONAL FACTORS LIQUID MEDIA AEROBIC MEDIA SIMPLE MEDIA SEMISOLID MEDIA ANAEROBIC MEDIA COMPLEX MEDIA SOLID MEDIA SYNTHETIC MEDIA SPECIAL MEDIA CLASSIFICATION
- 11. SPECIAL MEDIA A. ENRICHED MEDIA B. ENRICHMENT MEDIA C. SELECTIVE MEDIA D. DIFFERENTIAL MEDIA F. TRANSPORT MEDIA
- 12. PHYSICAL TYPE CULTURE MEDIA Solid media – contains 2% agar Colony morphology, pigmentation, hemolysis can be appreciated. Eg: Nutrient agar, Blood agar Liquid media – no agar. o For inoculum preparation, Blood culture, for the isolation of pathogens from a mixture. o Earliest liquid medium: urine or meat broth used by Louis Pasteur Eg: Nutrient broth Semi solid medium – 0.5% agar. Eg: Motility medium
- 14. Simple or basal media are culture media which contain the minimum adequate nutrition for non fastidious organisms Example:- Nutrient broth/agar Most common in routine diagnostic laboratories Eg: Nutrient Broth, Nutrient Agar NB consists of peptone, beeft extract, NaCl, water • NB + 2% agar = Nutrient agar • Agar conc. Reduced (0.2 - 0.5%) = Semi-solid medium Uses:- 1. This is basis of most of the media used in the study at common pathogenic bacteria. 2. It is used for subcultures of certain organisms. 2. BASED ON NUTRITIONAL FACTORS A. Simple media Nutrient broth Nutrient agar
- 15. B.. COMPLEX MEDIA Media that contain some ingredient of unknown chemical composition (exact chemical composition not known ). Complex media may provide complete nutritional requirement of many different microorganism( fastidious) whose nutritional requirement not known. They have added ingredients. Provide special nutrients It usually contain complex material of biological origin etc blood, milk, peptone, beef extract, yeast extract. Eg; Nutrient broth, Tryptic soya broth, Mac Conkey Agar, soil extract agar, Oat meal agar
- 16. C. Synthetic or defined media Defined media are often used to culture photolithotrophic autotrophs such as cyanobacteria and photosynthetic protists. They can be grown on relatively simple media containing CO2 as a carbon source (often added as sodium carbonate or bicarbonate), nitrate or ammonia as a nitrogen source, sulfate, phosphate, and a variety of minerals. Example :- Dubo’s medium Media prepared from pure chemical substances and its exact composition is known. Defined media are used widely in research, as it is often desirable to know what the experimental microorganism is metabolizing.
- 17. D. SEMIDEFINED MEDIA In these media the exact chemical composition of the constituents is not known because substances like meat and peptone are used. Eg: peptone water – 1% peptone + 0.5% NaCl in water Most of the culture media used for routine diagnostic work are semidefined culture media. SPECIAL MEDIA (FUNCTIONAL TYPE OF 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. Examples:- Blood agar, Chocolate agar
- 18. TYPE : Enriched media.; APPEARANCE : Red color; COMPOSITION : Sterile Nutrient agar + Defibrinated sheep blood USES : Routine culture Widely used in medical bacteriology It is also an indicator medium showing the haemolytic properties of bacteria such as Streptococcus pyogenes. BLOOD AGAR
- 19. Choclate Agar Media TYPE : Enriched media.; Appearance : Chocolate brown color. Melt the desired amount of nutrient agar. Cool it in a water – bath at 75º C . Add 10 ml of sterile blood . Allow the medium to remain at 75º C. Mixing the blood and agar by gentle agitation from time to time until the blood become chocolate brown in color, within about 10 min. USES CULTURE OF Neisseria CULTURE OF Haemophilus influenzae CULTURE OF Pneumococcus - to supply blood factors like X factor (Hemin) and Y factor (NAD)
- 20. Enrichment media In this media nutritional environment is adjusted such a manner as to enhance selectivity the growth of certain bacterial type in a given mix population.eg extract of plant and animal tissue in NB &NAM Liquid media used to isolate pathogens from a mixed culture. Media is incorporated with inhibitory substances to suppress the unwanted organism. Eg: Selenite F Broth – for the isolation of Salmonella, Shigella – It is enrichment medium for culture of Salmonella typhi and paratyphi bacilli from stool sample Principle:- at neutral pH solution acid salinity has high toxicity to coli form group of bacteria and not to most of the salmonella groups. Alkaline Peptone Water – for Vibrio cholerae Selenite F Broth Alkaline Peptone Water
- 21. C. Selective media It is a medium favor the growth of particular microorganisms in which certain substances are present which inhibit all other bacteria except the desired bacteria. It encourages the growth of particular species from a mixed inoculum. Bile salts or dyes like basic fuschsin and crystal violet favor the growth of gram-negative bacteria by inhibiting the growth of gram-positive bacteria; the dyes have no effect on Gram negative organisms. The inhibitory substance is added to a solid media. Endo agar, eosin methylene blue agar, and Mac Conkey agar are three media widely used for the detection of E. coli and related bacteria in water supplies and elsewhere. Eg: • Mac Conkey’s medium for Gram negative bacteria Eosine methylene medium for Gram negative bacteria • Thiosulfate Citrate Bile Salts Sucrose(TCBS) – for V. cholerae • Lowenstein Jensen (LJ medium) – M. tuberculosis • Wilson and Blair medium – S. typhi • Potassium tellurite medium – Diphtheria bacilli
- 22. Example:- TCBS -It is light green translucent medium kept in petridish -It is selective medium for Vibrio cholera -Principle:- Bile salt inhibit the growth of normal commensals (unwanted bacteria). Vibrio chloerae produce acid by fermentation of sucrose which acts on bromothymol blue (indicator) producing yellow colonies. Example:- EMB agar media •selective for gram negative bacteria •The dye methylene blue in the medium inhibit the growth of gram positive bacteria Example:- Lowenstein –Jenson medium • is solid medium used for Mycobacterium tuberculosis. • contain penicillin, nalidixic acid and malachite green to inhibit growth of gram positive and gram negative bacteria, • in order to limit growth to Mycobacteria species only
- 23. Potassium Tellurite media Potassium Tellurite act as selective agent and Inhibit the growth of most Gram positive and Gram negative bacteria except Corynebacteria diphtheriae Used for isolation of Corynebacteria diphtheriae Reduce Potassium Tellurite to tellurium and produce gray black colour colony Direct detection from nose and throat by using swabe Wilson-Blair medium contain an indicator which changes its colour when a bacterium grows in them. The black colour of colonies is due to the ability of these organisms to reduce bismuth sulphite to sulphide in the presence of glucose coliforms are inhibited by brilliant green and bismuth sullphite For isolation of Salmonella typhi and S. paratyphi – S. typhi forms black colonie
- 24. . Differential media Differential media are media that distinguish among different groups of microbes and even permit tentative identification of microorganisms based on their biological characteristics. A media which has substances incorporated in it enabling it to distinguish between bacteria. Blood agar is both a differential medium and an enriched one. It distinguishes between hemolytic and non-hemolytic bacteria. Hemolytic bacteria (e.g., many streptococci and staphylococci isolated from throats) produce clear zones around their colonies because of red blood cell destruction Eg: Mac Conkey’s medium (both a differential and selective medium) Peptone Lactose Agar Neutral red Taurocholate MacConkey agar is a culture medium designed to grow Gram-negative bacteria. It is a useful medium for the cultivation of enterobacteriacea in water and biological specimens. Distinguish between lactose fermenters & non lactose fermenters.
- 25. MacConkey agar showing both lactose and non-lactose fermenting colonies. Lactose fermenting colonies are pink whereas non-lactose fermenting ones are colourless or appear same as the medium It contains lactose and neutral red to distinguish the lactose- fermenting coliforms from the lactose non –fermenting salmonella and shigella groups. It contains Bile salts to inhibit non-intestinal bacteria and most Gram-positive bacteria, except Enterococcus and some species of Staphylococcus i.e. Staphylococcus aureus. Neutral red dye : which stains microbes fermenting lactose. Crystal violet dye : which also inhibits certain Gram-positive bacteria).
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- 27. Gram-negative bacteria growing on the media are differentiated by their ability to ferment the sugar lactose. Lactose fermenter cause the pH to drop and is detected by neutral red, (red at pH's below 6.8.) which appear as bright pink to red colonies on the agar. Uses Acting as a visual pH indicator, the agar distinguishes those Gram-negative bacteria that can ferment the sugar lactose (Lac+) from those that cannot (Lac-). By utilizing the lactose available in the medium, Lac+ bacteria such as Escherichia coli Enterobacter spp. Klebsiella spp. will produce acid, which lowers the pH of the agar below 6.8 and results in the appearance of red/pink colonies
- 28. It is a valuable non-inhibitory growth medium used in the isolation and differentiation of urinary organisms. Being electrolyte deficient, it prevents the swarming of Proteus species (Proteus mirabilis and Proteus vulgaris are frequently involved in urinary tract infection Cystine Lactose Electrolyte Deficient medium(CLED Serretia
- 29. . Transport media Media used for the temporary storage transporting the samples . Minimize bacterial outgrowth from time of c ollection to time its received at the laboratory to processed. Delicate organisms may not survive the time taken for transporting the specimen without a transport media. Such media ideally maintain the viability of all organisms in the specimen without altering their concentration. Transport media typically contain only buffers and salt. The lack of carbon, nitrogen, and organic growth factors prevents microbial multiplication. Eg: Stuart’s medium – non nutrient soft agar gel containing a reducing agent to prevent oxidation and charcol to neutralize bacterial inhibitor for gonococci. Buffered glycerol saline – Used to transport feces suffering from bacillary dysentery caused by enteric bacilli
- 30. Anaerobic media These media are used to grow anaerobic organisms. Semisolid media with reducing agent sodium thioglyclate and cystine added to provide reduce environment Eg: Robertson’s cooked meat medium, Thioglycolate medium.