Lab 2. Microbiological Culture Media PDF

Basic Microbiology Practical No.: 22(Online) Microbiological Culture Media Intended learning Objective (ILOs): By the end of this lab the student will be able to: 1. Know the importance of sterile conditions and aseptic techniques 2. Explain t...

Basic Microbiology Practical No.: 22(Online) Microbiological Culture Media Intended learning Objective (ILOs): By the end of this lab the student will be able to: 1. Know the importance of sterile conditions and aseptic techniques 2. Explain the purpose and preparation of different media. 3. Know the following commonly used culture media as well as their uses in a clinical microbiology laboratory. a. Basic medium b. Differential Medium c. Selective medium d. Enriched and e. Enrichment media Precaution: 1. Wear appropriate personal protective equipment for the laboratory procedure. 2. Maintain aseptic condition 3. Select standard setting of autoclave (No over or under sterilization) Page 1 Introduction/Background: q Microorganisms are defined as organisms that cannot be seen by the unaided eyes that are distributed widely in nature and on/in human body. q Most of the microorganisms on our body are harmless which known as normal flora. However, some can cause diseases and these are called pathogens. q These microorganisms require different types of nutrients which serve as their source of energy q Each microorganism also requires certain environmental conditions in order to grow and reproduce. q In microbiology, there are different types of media that is used for isolation of different types of organisms. q Microbiology culture media is basically a calculated mixture of all necessary nutrients required to grow/isolate specific microorganism/s, which can be either liquid (Broth) or solid (Agar). Depending on the type and combination of nutrients, different categories of media can be made. q Maintaining of Sterile condition is very crucial when isolating a specific organism. Therefore, various aseptic techniques are used. The most common growth media for microorganisms are Nutrient broth (No agar) and Nutrient Agar (contain 1.5% agar per weight) is the basic microbiological growth medium that is commonly used for the routine cultivation of non-fastidious bacteria. v Specialized media are sometimes required for microorganism and cell culture growth. Page 2 TYPES OF MEDIA 1. Solid media: v Liquid media are often mixed with agar and poured into Petri dishes to solidify. These agar plates provide a solid medium on which microbes may be cultured. v Agar is particularly used as a solidifying agent because it will not melt until the medium is heated to near boiling(95°C-100°C), but will remain melted until cooled to around 42°C. v After sterilization, the liquified agar medium is poured into sterile Petri plates and let to cool down and solidify. Petri plates are commonly made of plastic, and already sterilized. v Agar plates allow us to isolate bacterial colonies or count them depending on the culture technique that is used. v Used widely for the isolation of pure cultures. v The usual solidifying agent for solid or semisolid medium is agar Page 3 2. Liquid media: v Nutrient broth does not contain agar. v Nutrient Broth are used for enrichment of specified bacteria like E. coli and salmonella Broth culture: Bacteria or any other growing microorganism in liquid nutritional medium (broth) in test tube is known as broth culture Page 4 TYPES OF GROWTH MEDIA: 1. Nutrient media: v A source of amino acids and nitrogen (e.g., beef, yeast extract). Nutrient media contain all the elements that most bacteria need for growth and are non-selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory culture collections 2. Minimal media: v It contain the minimum nutrients possible for colony growth, generally without the presence of amino acids, and are often used by microbiologists and geneticists to grow "wild type" microorganisms 3. Selective media: v Used for the growth of only select microorganisms. e.g.: Eosin methylene blue (EMB), MacConkey(MAC), Mannitol salt agar (MSA), Xylose lysine deoxyscholate (XLD), Blood agar & Chocolate agar 4. Differential media v It is used for the detection of microorganisms and by molecular biologists to detect recombinant strains of bacteria. v Differential media or indicator media distinguish one microorganism type from another growing on the same media. v Some media are selective and differential at the same time Examples of differential media include: Page 5  a. Eosin methylene blue (EMB) which is differential for lactose and sucrose fermentation b. MacConkey (MAC), which is differential for lactose fermentation c. Mannitol salt agar (MSA), which is differential for Mannitol fermentation 5. Transport media: v Solution of buffers with some nutrients but no growth factors. v Used to preserve the viability of the bacteria during transportation while preventing multiplication. v The main aim is to maintain the specimen as near its original state as possible. v Ex. Thioglycollate broth for strict anaerobes 6. Enriched media: v Commonly used to harvest as many different types of microbes as are present in the specimen. Ex. Blood agar & Chocolate agar v Also used for some fastidious microorganisms Page 6 Part One: PREPARATION OF MEDIA PRINCIPLE: The survival and growth of microorganisms depend on available and favorable growth environment. Culture media are nutrient solutions used in laboratories to grow microorganisms. PROCEDURE: Equipment: Safety cabinet, Bunsen burner, pH meter, dispenser, magnetic stirrer, Bunsen burner and autoclave. Materials: Measuring cylinder, flask, cotton, gloves, Petri plates, test tubes. Reagents: Bacteriological chemicals or Base of media, Distilled water, pH indicator paper. Method: The basic steps for preparing the culture medium are listed below: (NOTE: You need to calculate how much is required of each component depending on the final volume) 1. Weigh required media 2. Select a container twice the size of the final volume of distilled water. Add approximately 90% of the final volume of water. 3. While stirring the water add the powdered medium and stir until completely dissolved. 4. Add desired heat stable supplements (e.g. sucrose, gelling agent, vitamins, auxins, cytokinins, etc.) 5. Add additional distilled water to bring the medium to the final volume. Page 7 6. While stirring, adjust medium to desired pH using NaOH, HCl, or KOH, if needed. 7. If a gelling agent is used, heat until the solution is clear. 8. Dispense the medium into the culture vessels before (or after) autoclaving according to your application. Add heat labile constituents after autoclaving. 2 9. Sterilize the medium in a validated autoclave at 1 kg/cm (15 psi), 121°C, for the time period described under Sterilization of Media. (Note : few medias cannot be autoclaved read the instructions on the media bottle before autoclave. Other methods will be used for sterilization) 10. Allow medium to cool prior to use (Why?). *Heating may be required to bring powders into solution. 11. In case of enriched media preparation, sterile blood or, serum or egg is always added after sterilization of the media. Page 8 Preparation of some media: Ø NUTRIENT AGAR: Nutrient broth: Its does not contain agar. Requirements: Equipment: Conical flask, measuring cylinder, Weighing Balance, Hot plate stirrer, Autoclave, Oven. Supplies: Dehydrated media, Distilled Water, Sterile petri dishes Procedure: v 28g Nutrient Agar in 1000ml distilled water heat to boiling to dissolve the medium completely. v Sterilize by autoclaving at 15lbs pressure, 121°C temperature for 15minutes. v Cool to 45-50°C and pour into sterile Petri plates. v The surface of the medium should be dry when inoculated. Page 9 Nutrient agar typically contains (w/v): 1. 0.5 % peptone , 2. 0.3 % beef extract, 3. 1.5 % agar, 4. pH adjusted to neutral at 25°C Uses: They are used for the general cultivation and maintenance of bacteria kept in laboratory culture collections. q Nutrient broth is made identically, except omitting the agar. Ø MacConkey agar It contains: i. Bile salts to inhibit most Gram-positive bacteria, except Enterococcus and some species of Staphylococcus i.e. Staphylococcus aureus, ii. Crystal violet dye: It is also inhibits certain Gram-positive bacteria. Page 10  iii. Neutral red dye which stains microbes fermenting lactose (pH indicator). iv. Lactose and peptone. Procedure: v 55g MA in 1000ml distilled water heat to boiling to dissolve the medium completely. v Sterilize by autoclaving at 15lbs pressure, 121°C temperature for 15minutes. v Cool to 45-50°C and pour into sterile Petri plates Uses: v For the selective isolation, cultivation and differentiation of coliforms and enteric pathogens based on the ability to ferment lactose. v Lactose fermenting organisms appear as red to pink colonies. v Lactose non-fermenting organisms appear as colorless or transparent colonies Growth of some microorganisms: i. Escherichia coli: Pink colonies (lactose fermentation) ii. Proteus mirabilis: Colorless colonies, no spreading iii. Enterococcus sp.: No growth v Incubation conditions: Aerobic at 37° C for 24 hrs. Page 11 Ø C.L.E.D AGAR v C.L.E.D Agar (Cystine Lactose Electrolyte Deficient Medium) is a valuable non-inhibitory growth medium used in the isolation and differentiation of Urinary tract Organisms. Procedure: v See the directions mention on bottles and follow the same procedure as above. Uses: For Urine culture isolation. Lactose fermenters produce yellow colonies & non-lactose fermenters appear blue. Page 12 Ø XLD AGAR: XLD Agar (Xylose lysine desoxyscholate), which is selective for Gram-negative bacteria Procedure: v See the directions mention on bottles and follow the same procedure as above Uses: v Isolation of Salmonella and Shigella species from clinical samples and from food Salmonella sps: red colonies, some with black centers. Shigella species: red colonies. Coliforms: yellow to orange colonies. Pseudomonas aeruginosa: Pink, flat, rough colonies Page 13 Ø BLOOD AGAR: v It Contains mammalian blood (usually sheep or horse), typically at a concentration of 5–10%. Procedure: v 40g Blood agar base in 1000ml distilled water heat to boiling to dissolve the medium completely. v Sterilize by autoclaving at 15lbs pressure, 121°C temperature for 15minutes. v Cool to 45-50°C, v Add 5-10% of mammalian blood and pour into sterile Petri plates. Uses: For the isolation, cultivation and detection of hemolytic activity of streptococci, pneumococci and other particular fastidious microorganisms. Control organisms: i. Streptococcus pneumoniae : Good growth, Alpha - hemolysis , ii. Streptococcus pyogenes : Good growth, Beta-hemolysis Blood agar plates are often used to diagnose infection v α-hemolysis will only partially lyse (the cells are either lysed or not- it is the digestion that may be incomplete) the hemoglobin and will appear green. (Ex. Streptococcus pneumoniae). Page 14  v β-hemolysis activity will show lysis and complete digestion of red blood cell contents surrounding colony. (Ex. Streptococcus pyogenes). v γ-hemolysis (or non-hemolytic) is the term referring to a lack of hemolytic activity. Ø Chocolate Agar: v It is also contains heated mammalian blood (usually sheep or horse), typically at a concentration of 5–10%. Procedure: v 40g Blood Agar in 1000ml distilled water heat to boiling to dissolve the medium completely. v Sterilize by autoclaving at 15lbs pressure, 121°C temperature for 15minutes. Page 15  v Add 5-10% of mammalian blood at 80-85°C (Blood cells have been lysed by heating the cells to 56 °C). v Cool to 45-50°C and pour into sterile Petri plates Uses: For the isolation and cultivation of a variety of fastidious microorganism, such as respiratory bacteria - Haemophilus influenzae. Note: It is a type of blood agar. No chocolate is actually contained in the plate; it is named for the coloration only. Control organisms: v Hemophilus influenzae and Neisseria gonorrhoeae : Good growth Ø Sabouraud Dextrose Agar (SDA): v It is used to cultivate dermatophytes and other types of fungi, and can also grow filamentous bacteria such as Nocardia. Procedure: v See the directions mention on bottles and follow the same procedure as above Page 16 Uses: For the cultivation of pathogenic and nonpathogenic fungi, especially dermatophytes. Control organisms: i. Aspergillus flavus, Candida albicans: Growth ii. Streptococcus pneumoniae: No growth Ø MÜLLER-HINTON AGAR (MHA): Procedure: v 38g MHA in 1000ml distilled water heat to boiling to dissolve the medium completely. v Sterilize by autoclaving at 15lbs pressure, 121°C temperature for 15minutes. v Cool to 45-50°C and pour into sterile Petri plates Uses: For antimicrobial susceptibility testing of a variety of non- fastidious, rapidly-growing microorganisms. v It is also used to isolate and maintain Neisseria and Moraxella species. Control organisms: i. E.coli – ATCC N0. 25922 Page 17  ii. S. aureus – ATCC N0. 25923 iii. P. aeruginosa – ATCC N0. 27853 v Growth, Inhibition Zone followed Standard interpretative charts References: 1. Bharti Arora, D. R. Arora (2015): Practical Microbiology. ISBN: 81-239-14095-9. Page 1 – 218. 2. Steven Obenauf, Susan Finazzo (2016): Microbiology Fundamentals. A Clinical Approach. Page 1 – 318. Page 18