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  INTRODUCTION OF CULTURE MEDIA Most bacteria can be cultured artificially on culture media containing required nutrients, pH and osmotic pressure. The microorganisms grow in an atmosphere and temperature most suited to their metabolic reactions. The pathogens are isolated in pure culture so that they can be identified and tested for their sensitivity to antimicrobials. The specimens are cultured in known volumes, and the number of bacterial colonies appearing after incubation can be counted. Operation room requirements and blood from blood bank are frequently checked for sterility by using pure culture methods. Vaccines and antitoxins require the growing of bacteria under controlled conditions. Stock cultures are also useful for the teaching institutes for practical training purposes. COMPOSITION OF CULTURE MEDIA The basic ingredients, which are common to the many ofthe frequently used media are as follows: 1.     Water:It allows fluids to enter and leave cells more readily and to enhance thechemical reactions. 2.     Sodiumchloride: Presence of sodium chloride maintains the isotonicityof bacterial cells. 3.     Peptones:It is a source of readily available nitrogen. 4.     Buffers:They maintain a constant pH in culture media. 5.     Indicators:In culture media the indicators are useful in the detection of acid or alkaliproduction by microorganisms. Indicators such as phenol red, methyl red andBromocresol purple are used to adjust pH of the culture media. 6.     Solidifyingagents: Agar, gelatin, egg yolks and serum are used assolidifying agents of the culture media. 7.     Selectiveagents: These are special chemicals introduced into theculture media for inhibiting some types of bacteria, while allowing otherbacteria to grow. Examples: (a) Crystal violet inhibits Staphylococci but nottubercle bacilli (b) 6.5% (w/ v) sodium chloride is inhibitory to mostStreptococci but not S. faecalis. 8.     Additivefor enrichment: The substances such as sheep blood, horse blood,rabbit serum or calf’s ground hearts allow fastidious organisms to grow sincethese organisms, may not survive in ordinary culture media. 9.     Reducingsubstances: The substances such as thioglycollate are used toremove free oxygen from the medium for the growth of anaerobic bacteria.    THEDIFFERENT TYPES OF CULTURE MEDIA Basic Media:-These support the growth of microorganisms that do nothave special nutritional requirements. They are often used (a) To maintainstock cultures of control strains of bacteria and (b) For subculturingpathogens from selective media prior to performing biochemical and serologicalidentification tests. Examples: (1) Nutrient agar (2) Nutrient broth. Enriched Media:- These are enriched with (a) Whole blood (b) Lysed blood (c) Serum (d) Extra peptones and (e) Vitamins to support the growth of particular pathogens such as Hemophilus influenzae, Neisseria and Streptococcusspecies. Examples: (I) Blood agar (II) Tryptone soya media. Selective Media:- These media contain substances that accelerate the  growth of required pathogens only and prevent or slow down the growth of other  microorganisms. Example: XLD agar: It is used for the growth of Salmonellae and Shigellae. The bile salts present in this media inhibit the growth of many fecal commensals. Differential (Indicator) Media:- These contain indicators, dyes or other substances which help to differentiate microorganisms. Example: TCBS agar contains the indicator bromothymol blue which differentiates sucrose fermenting from non-sucrose fermenting vibrio species. Transport Media:- When specimens are not cultured soon after collection,to prevent overgrowth and also to ensure survival of pathogens, transport mediaare used. These are mainly used to transport microbiological specimens fromhealth centers to the district pathological laboratories. Example: (1) Amiestransport medium (2) Cary Blair medium. DIFFERENT FORMS OF CULTURE MEDIA 1.      Solid Culture Media:- This isused in petri dishes and in test tubes (slope cultures) and prepared by addingSolidifying agent  (1.0 – 1.5%) (w/v).Microorganisms grow on this and form colonies after multiplication. This helpsto identify the organism. 2.      Semisolid Culture Media :- This isprepared by adding Solidifying agent (0.4-0.5%) (w/v) to a fluid medium. Theseare used mainly as transport media and for the testing of motility of theorganisms. 3.      Fluid Culture Media:- These mediaare mainly used as biochemical testing media, blood culture media or theenrichment media. PREPARATION OF CULTURE MEDIA  Mostculture media are available commercially in readymade dehydrated form. It isless costly to use readymade media, since the ingredients are often required insmall amounts but available in large quantities if purchased. Some of thechemicals are also difficult to obtain. To ensuregood performance and reproducibility in the results the following must beperformed correctly- 1.    Weighing and dissolving of theingredients 2. Addition of heat sensitive material 3.  pH testing 4. Dispensing and sterilization 5. Sterility testing and qualitycontrol 6.storage  Note 1.      The heat sensitive ingredients such as blood or serum should be brought to room temperature and added when the medium has cooled to about 50°C. 2.      A fluid medium should be tested for accurate pH by using a narrow, range pH paper. 3.      For sterilizing culture media, it is necessary to use manufacturer’s instructions. The commonly used methods for sterilization are- a) Autoclaving (b) Steaming at 100°C and (c) Filtration. It is necessary to use correct temperature and correct length of time. Precautions ·  Autoclaving is used to sterilize most agar and fluid media. · Steaming at 100°C: Media such as Cary Blair transport medium contain ingredients that would break down above 100°C. Steaming can be performed in an autoclave with a loose lid. ·   Filtration: Serum and solutions containing carbohydrates, urea, etc. are heat sensitive and hence cannot be autoclaved. Hence, the media containing such substances are filtered to remove bacteria. ·  Sterility testing: Media in tubes and bottles: Incubate the entire batch at 37°C overnight. Contamination is indicated by appearance of turbidity in a fluid medium and growth on a solid medium. · Control of media: Appropriate control species are used to inoculate slants or plates of the medium (quarter part). After overnight incubation, the cultures are examined for (a) Degree of growth (b) Size of colonies and (c) Other characteristics. ·  Storage of culture media: Dehydrated culture media and dry ingredients (agars, peptones, bile salts, etc.) can be stored at room temperature (25°C ± 5°C) in a cool and dry place, away from direct light. Additives such as blood, serum, urea and carbohydrates in solution form are stored at 2-8°C in the refrigerator. Basic Method of Preparation of Culture Medium – Step by Step 1.      Weigh the solid ingredients by using an analytical balance. 2.      Add sufficient quantity of distilled water. 3.      Dissolve the

1. Nutrient Agar Nutrient agar is a solid medium used in microbiology to grow a wide range of non-fastidious organisms, including bacteria and fungi. Composition of Nutrient Agar: Component Amount Peptone 0.5% Beef Extract 0.3% Sodium Chloride 0.85% Agar 1.5% Water 1000 ml 2. Blood Agar It is a general purpose, enriched and differential solid medium, which supports the growth of most ordinary bacteria. It is also useful to detect and differentiate hemolytic bacteria, especially Streptococcus species.Blood supplies a number of required nutrients for the growth of fastidious organisms. Composition of Blood Agar: Component Amount Nutrient agar 500 ml Sterile Defibrinated blood 25 ml Procedure: Transfer sterilized nutrient agar to 50°C water bath. When it is cooled to 50°C, add aseptically sterile defibrinated sheep or horse blood. Mix gently and dispense aseptically in sterile petri dishes. pH of the medium should be adjusted to 7.3.               Blood should be free from hemolysis. Sheep or rabbit blood can be used for most of the pathogens. Use of human blood should be avoided, since certain substances in human blood may be inhibitory to the growth of certain pathogens such as hemolytic Streptococci. Sheep blood inhibits the growth of Haemophilus haemolyticus (human throat commensal).       Note: 3. Chocolate (heated blood) agar It is used to grow Haemophilus influenzae and other pathogens which require highly nutritious medium, such as Neisseria meningitidis and Streptococcus pneumoniae. Formula and preparation: It is prepared by using the same procedure used for the preparation of blood agar except after adding blood, the medium is heated at 70°C in a water bath until it becomes brown in color. The medium should be mixed gently for about 10–15 minutes. Allow it to cool to about 45°C. Dispense aseptically in sterilized petri dishes. Note: The medium should not be overheated. The duration of heating should not be prolonged.   4.    Crystal violet blood agar This is used as a selective medium for Streptococcus pyogenes. • Principle:- At a concentration of 1:500,000 crystal violet inhibits the growth of Staphylococcus aureus and retards the growth of commensals (throat specimen). • Formula and preparation 1. Sterile blood agar:                       100 ml. 2. 0.02% (w/v) crystal violet:         1.0 ml. Mix well and dispense aseptically in sterile petri dishes. 5. Neomycin Blood Agar This is used as a selective medium for obligate anaerobes. Principle: Facultative anaerobic Gram-negative rods are inhibited at a concentration of 70 µg/ml neomycin sulfate. Formula and Preparation a) Prepare stock neomycin sulfate (70,000 µg/ml) Neomycin sulfate    →     0.5 g Sterile water             →      5 ml (This contains 0.35% neomycin base) b) Preparation of working neomycin (37500 µg/ml) Add 2 ml of 70,000 µg/ml stock neomycin solution to 8 ml sterile water. Blood agar               →        250 ml Working neomycin →        1.0 ml Mix well and pour aseptically in sterile Petri dishes.Concentration of solution C = 700 µg/ml neomycin/ml. 6. Brain Heart Infusion Broth and Agar It is an enriched medium used for the growth of fastidious bacteria, yeasts, and molds. Preparation of BHI (Liquid Medium) I. Beef heart infusion        →           250.0 gII. Calf brain infusion        →           200.0 gIII. Proteose peptone        →          10.0 gIV. Dextrose                        →          2.0 gV. Sodium chloride            →          5.0 gVI. Disodium phosphate  →          2.0 gVII. Distilled water            →          1000 ml Dissolve the contents in 800 ml of distilled water and make the final volume to 1000 ml. Sterilize the medium by autoclaving at 121°C for 15 minutes. Dispense aseptically after cooling the agar medium to 45–50°C. Note: With the addition of 1.5% agar, BHI agar can be used as a solid medium. 7. Bordet Gengou’s Medium It is a selective medium used for the isolation of the causative agent of whooping cough, Bordetella pertussis. Formula and Preparation I. Potato infusion          —              125.0 gII. Peptone                     —               10.0 gIII. Sodium chloride      —               5.0 gIV. Glycerol                     —               20.0 gV. Agar                            —               30.0 g Procedure Mix potato with glycerol and about 100 ml of distilled water; boil and strain through gauze. Add the filtrate to the mixture of other ingredients and keep for 5 minutes. Boil the solution to dissolve the agar. Make the final volume 1000 ml. pH of the medium should be adjusted to 6.4–6.6. Sterilize the medium in 100 ml volumes in bottles or flasks. Sterilize the containers by autoclaving at 121°C for 15 minutes. Store at 2–8°C. 8. Brilliant Green Agar This medium is used for the selective isolation of Salmonella. Formula and Preparation I. Proteose peptone  —               10.0 gII. Yeast extract          —               3.0 gIII. Sodium chloride   —               5.0 gIV. Lactose                  —              10.0 gV. Sucrose                   —              10.0 gVI. Phenol red             —              0.08 gVII. Brilliant green      —               0.0125 gVIII. Agar                 

It demonstrates the presence of metachromatic granules found in Corynebacterium diphtheriae. Smears of throat swab are useful in the diagnosis of diphtheria by Albert’s staining method. Reagents 1. Albert’s solution ‘A’ a) Add 1.0 ml of glacial acetic acid to 99 ml of distilled water. b) Add 0.15 g of toluidine blue and 0.2 g of malachite green in 2.0 ml of (95%) ethyl alcohol by grinding in a mortar. c) Mix solution (a) and solution (b). Keep for 24 hours and afterwards filter it and store in an amber colored dropping bottle. 2. Albert’s solution ‘B’ a) Add 3.0 g of potassium iodide in 100 ml of distilled water. b) Add 2.0 g of iodine in solution (a). Mix thoroughly and store in an amber colored dropping bottle. Stability of the Reagents The reagents are stable at room temperature (25°C ± 5°C). Procedure Flood the heat fixed smear with Albert’s staining solution ‘A’ for 5 minutes. Drain the staining solution but do not wash. Flood with solution ‘B’ and keep for 1 to 2 minutes. Wash carefully under running tap water, drain, blot dry and examine under oil immersion objective. Results Metachromatic granules: Bluish black. Bacillary body: Green or bluish green.

Simple Staining Simple staining is used to stain prokaryotic cells, e.g., microorganisms, to determine the shape, size, and arrangements. In this staining method, only a single dye is needed. It’s simple with the procedure which includes covering fixed smears with stain for a minute, and excess stain is washed off with water and blotted dry. In this case, basic dyes are used which are methylene blue, crystal violet, and carbolfuchsin. (a) Crystal violet stain of Escherichia coli(b) Methylene blue stain of Corynebacterium