CLINICAL BACTERIOLOGY LABORATORY CULTURE MEDIA OUTLINE • Culture Media o Introduction o Nutrients o Oxygen o Moisture o pH o Temperature • Preparation of Culture Media o Culture Media Preparation o Sterile Conditions & Autoclaving • Culture Media o 3 Types of Culture o Classification of Culture Media • Inoculation Techniques o Aseptic Technique o Materials o Procedures CULTURE MEDIA INTRODUCTION • Microorganisms depend on a number of factors such as nutrients, oxygen, moisture and temperature to grow and divide. In the laboratory, except for the above factors, the culture medium should be sterile and contamination of culture with other organisms should be prevented. Let us briefly discuss a few of the more important factors for the growth of microorganisms. NUTRIENTS • A microbiological culture medium must contain available sources of hydrogen donors and acceptors, carbon, nitrogen, sulfur, phosphorus, inorganic salts and, in certain cases, vitamins or other growth-promoting substances. These were originally supplied in the form of meat infusions that were, and still are in certain cases, widely used in culture media. Beef or yeast extracts can replace meat infusions. The addition of peptone provides a readily available source of nitrogen and carbon. • Peptone is used in culture media to mainly supply nitrogen. Most organisms are capable of utilizing the amino acids and other simpler nitrogenous compounds present in peptone. Thus, in many cases, the complicated infusion media can be replaced by simpler media prepared by using the proper peptones in place of the meat infusions. Certain bacteria require the addition of other nutrients, such as serum, blood, etc. to the culture medium upon which they are to be propagated. Carbohydrates may also be desirable at times, and certain salts such as calcium, manganese, magnesium, sodium, and potassium seem to be required. Dyes may be added to media as indicators of metabolic activity or for their selective inhibitory powers. Growth promoting substances of a vitamin-like nature are essential or assist greatly in the development of certain types of bacteria. Many of these substances are given for individual bacteria in Bergey's Manual of Determinative Bacteriology (Incidentally, this is a reference text that you should familiarize yourself with when working with microorganisms.) OXYGEN • Most bacteria are capable of growth under ordinary conditions of oxygen tension. Certain types, however, are capable of deriving their oxygen from various substrates. The aerobic organisms require the free admission of air, while the anaerobes grow only in the absence of atmospheric oxygen. Between these two groups are the microaerophiles that develop best under partial anaerobic conditions and the facultative anaerobes that develop under aerobic as well as anaerobic conditions. • It is easy to provide oxygen to aerobic and facultative anaerobic and even microaerophilic organisms; however, special gadgetry is required to exclude the atmospheric oxygen and provide an anaerobic condition. Such conditions are obtained by: o Addition of a reducing substance to the medium o Displacement of the air by carbon dioxide o Absorption of the oxygen by chemicals o Removal of oxygen by direct oxidation of readily oxidizable substances such as burning a candle, heating of copper, phosphorus or other readily oxidizable metals o Incubation in the presence of germinating grain or pieces of potato o Inoculation into the deeper layers of solid media, or under a layer of oil in liquid media or o A combination of the above methods. MOISTURE • Proper moisture conditions must prevail in the culture media for the growth of microorganisms. • A moist medium and atmosphere are necessary for the continued luxuriant growth of cells. • For example, if a medium in a plate is inoculated with an organism and wet cotton is placed in the plate and sealed, the organism will show profuse growth. The same organism might fail to show growth if the medium plate is not sealed and is too dry. pH • The pH of the culture medium, expressed as hydrogen ion concentration [H+], is extremely important for growth. • The majority of microorganisms prefer culture media that are approximately neutral, while others may require a medium that is distinctly acidic. TEMPERATURE • Every organism shows a rather general curve of growth as affected by temperature. Such a curve shows: o a minimum temperature below which growth stops, o an optimum temperature at which growth is luxuriant and o a maximum temperature above which the organism dies. Microorganisms are divided into three main groups (mesophilic, psychrophilic, and thermophilic) as far as optimum temperature requirements are concerned. • The usual range of temperatures suitable for the growth of mesophilic microorganisms lies between 15-43 °C. Psychrophilic microorganisms have, however, been known to grow and multiply at 0 °C.
• Thermophilic organisms may grow at temperatures even greater than 80 °C. In general, the pathogenic organisms have a temperature requirement of around 37 °C (body temperature) while saprophytes have a much broader latitude. L1: PREPARATION OF CULTURE MEDIA CULTURE MEDIA PREPARATION • Bacteria will grow on practically any source of organic food which provides carbon compounds to be respired for energy, and nitrogen compounds to be incorporated into proteins for growth. These substances are normally provided dissolved in water. However, in nature, bacteria can break down solid and insoluble substances by releasing enzymes into the substrate in which they are growing. These substances are thus broken down or digested to simpler substances and the process is called extracellular digestion because it takes place outside the bacterial cells. MATERIALS • Personal Protective Equipment • Aluminum foil • Autoclave • Dehydrated Culture Media • Distilled Water • Cotton plug/Cork • Analytical Scale/Triple Beam Balance • Spatula • Stirring Rod • Erlenmeyer Flask PROCEDURE • Plated Media o Make sure all the materials are dry. 1. Follow the directions written on the label of the dehydrated culture media. (This may vary per culture media). 2. Heat using an electric hot plate and gently stir until the agar mixture is completely dissolve. 3. Remove from hot plate and sterilize at 121OC for 15- 20 minutes. 4. Let it cool between 45-50OC. 5. Dispense approximately 20 mL onto petri dishes. Allow to solidify for 10-15 mins. 6. Label and do sterility testing. 7. Store at 2-8OC in an inverted position. • Tubed Media o Make sure all the materials are dry. 1. Follow the directions written on the label of the dehydrated culture media. (This may vary per culture media). 2. Using a clean pipette, distribute the agar mixture into several test tubes. 3. Sterilize at 121OC for 15-20 minutes. 4. Let it cool between 45-50OC. 5. Label and do sterility testing. 6. Store at 2-8OC. STERILE CONDITIONS & AUTOCLAVING • The media upon which microorganisms are grown must be sterile or free from all other forms of microbes. The usual method for sterilization of culture media is by means of the autoclave in which steam under pressure is the sterilizing agent. • Autoclave sterilization for 15 minutes at 15 pounds of pressure and at 121 °C is recommended for quantities of liquid media up to one liter (1 L). These settings are called the standard autoclaving conditions. If larger volumes are to be sterilized in one container, and if the medium is not hot when placed in the autoclave, a longer period should be employed. • The medium is prepared according to formula, distributed in tubes or flasks which are then plugged with non-absorbent cotton or loosely capped before being placed in the autoclave. • Plugs should fit neither too loosely nor too tightly. Screw cap tops or metal covers may also be used to close the tubes or flasks. Tubes should be placed in racks or packed loosely in baskets. Flasks should never be more than two-thirds full. • After the sterilization period has been completed, the source of steam is cut off and the autoclave is allowed to return to atmospheric pressure. Pressure should not drop too rapidly or the media will boil over, blowing the plugs from the tubes or flasks. Pressure should, however, drop rapidly enough to prevent excessive exposure of the media to heat after the sterilization period. CULTURE MEDIA (PPT) • Anything that possess nutritional and environmental requirements for bacterial growth • Culture - these are organisms obtained from a culture media
3 TYPES OF CULTURE • Pure Culture - one species • Mixed Culture - two ore more species • Stock Culture - pure spp. (used for research) CLASSIFICATION OF CULTURE MEDIA ACCORDING TO CONSISTENCY • Liquid Culture Medium - has no solidifying agent; 0% agar o E.g., Fluid Thioglycollate Medium, Nutrient broth, BHI, Selenite • Semi-Solid Medium - 0.5-1% agar o E.g., SIM (Sulfide Indole Motility) • Solid Medium o Liquefiable- contains 2-3% agar (e.g., EMB, MAC, BAP, CAP) o Non-liquefiable- Rice grain for fungi • Biphasic Medium - both solid and liquid media (e.g., Castanedas media for Brucella spp.) • *Agar- commonly used based media • Storage o Prepared Culture Media - refrigerated o Dry Culture Media - room temperature up to 30C ACCORDING TO COMPOSITION • Synthetic Culture Medium - commercially prepared media; exact composition is known • Non-Synthetic CM - ordinary broth; exact composition is unknown • Tissue Culture Medium - living cell media; virus isolation ACCORDING TO HOW THE MEDIUM IS DISPENSED • Plated Medium o Richard Petri - invented the petri dish • Tubed Medium o 3 Types of Tube Medium ▪ Slant ▪ Butt ▪ Slant/butt ACCORDING TO USE • Simple Medium/General Purpose Medium/General Isolation Medium - routine cultivation and maintenance; contains only necessary nutrition to support bacterial growth o E.g., Nutrient Agar, Nutrient Broth • Enrichment Medium - enhances the propagation of certain organisms o E.g., Selenite Broth, Tetrathionate Broth, Alkaline Peptone Water • Enriched Medium - contains nutritive supplements o E.g., BAP, CAP • Differential Medium - distinguishes organisms growing together by their differences in cultural characteristics o E.g., EMB, MAC, TCBS • Selective and Differential - E.g., EMB and TCBS o Eosin Methylene Blue ▪ Escherichia coli (Eosin Methylene Blue Agar) • *Green Metallic Sheen o MacConkey Agar o Thiosulfate Citrate Bile Salt Sucrose Agar • Selective Medium - promotes the growth of desired organisms but at the same time inhibiting the others o E.g., Lowenstein Jensen- for M. tb; inhibitor is malachite green; CAP with VCN antibiotics- selective for Neisseria ▪ Substances that inhibit gram (+) oraganisms: • Bile salts: Sodium desoxycholate • Dyes: Gentian Violet, Crystal Violet ▪ Substances that inhibit gram (-) organisms: • Potassium tellurite • Sodium azide
• Transport Medium - used for unexpected delay of processing o E.g., Stuart’s, Amie’s, CaryBlair, Transgrow • Media for Susceptibility o E.g., MHA (Mueller Hinton Agar) • Special/Specific Medium o Fletcher’s - Leptospira o Bordet-gengou - Bordetella pertussis o Lowenstein-Jensen/ Middlebrook 7H-10/Middlebrook 7H-9 broth - M. tuberculosis o Thayer-Martin - Neisseria group o MacBride Agar - Listeria monocytogenes o W Medium - Brucella o Mannitol Salt Agar - Staphylococci o Loeffler’s/Tinsdale/Tellurite - Corynebacterium diphtheriae L2: INOCULATION TECHNIQUES ASEPTIC TECHNIQUE • The use of aseptic technique is so that contamination is reduced or eliminated when plating organisms. It ensures that microbes are being handled in a safe manner to prevent contamination of the handler or others in the laboratory. And it means cleaning up after yourself so that no contamination remains after you have worked with bacteria or other microbes. It means being very careful with how you handle cultures, bacteria, and loops, treating the loops as sterile, and being careful not to touch anything with your loop prior to inoculation of the media, whether it be transferring a broth culture to a plate for streaking, or inoculating an isolated colony from a plate to another plate for purity and/or isolation. It may also mean inoculating many tubes of media and agar plates from a stock culture to identify a bacterium. It is crucial that you make sure that only your desired organism is successfully transferred during the inoculation process for successful and accurate identification. • Flaming the loop means to sterilize your loop or inoculating needle until is becomes bright red. The entire wire must be heated, but be careful not to insert the handle inside the incinerator or it might melt. • Disinfect your work area to kill any microbes that may be present. This process destroys vegetative cells and any viruses, but it may not destroy endospores. This is what the autoclave process is for. Cavicide wipes are used to wipe off countertop surfaces, along with bleach and 70% alcohol. • Loops and needles need to be sterilized before transferring any culture when using metal loops or inoculating needles to burn and destroy any contaminating organisms that might be present. If using plastic loops, ensure that they have not touched any surfaces or your lab coat prior to inoculation, and that the bottom of the bag or case that the loops are in is properly sealed. If you accidentally drop a loop prior to inoculation, throw it away in the biological waste bin and do not use it to inoculate your media. Take out a new loop. • Prior to inserting a cooled and sterilized loop or needle into a culture tube, once the cap is removed, flame the mouth of the tube. Inoculate your tube, reflame the moth, and recap it. Most tube media needs to have loosened caps prior to incubation. • After the inoculation is complete, you need to resterilize your loop or needle to destroy the organisms you just inoculated into your media. Return the loop or needle to its storage place and never place it down on the countertop surface. • Loops are used to inoculate or streak petri dish plates. The plate cover is raised and held at an angle over the plate to protect the surface from any contamination in the air, such as dust and bacterial particles. Never talk on the plates, as spit and organisms from your mouth may contaminate the cultures. Take care not to gouge or disturb the surface of the agar with the loop. • When all work for the day is complete, treat your work area with disinfectant and empty the biological waste bins and autoclave any bags and sharps containers that are full and ready for autoclaving. MATERIALS • Inoculating loop/needle • Bacterial culture/suspension • Alcohol lamp • Plated media • Tubed media PROCEDURES 1. Hold the inoculating loop or needle in a certain angle in the red heat of the Bunsen burner or alcohol lamp until it is red hot. 2. Ensure that the entire loop or needle receives adequate heating. 3. Let it cool. 4. Do not place the loop or needle on any surface of the countertop. 5. REMEMBER: Re-heat the loop or needle after use. PLATED MEDIA 1. Flame the inoculating loop red hot. 2. Using the left hand gently lift the lid of the agar, but continue to use it as a cover to prevent contamination. 3. Pick approximately 1-2 colonies using the inoculating loop or needle. 4. Carefully remove the loop/needle and hold it still as you replace the lid. 5. Inoculate/subculture on plated, tubed or broth media. 6. Flame the inoculating loop/needle red hot after its use. BROTH MEDIA 1. Heat the inoculating loop red hot. 2. Remove and hold the cotton plug with the little finger where your loop hand is. 3. Do aseptic technique by passing the lid of the tube through the flame. 4. Hold the tube at an angle the insert the loop. There should be a film of broth in the loop. Avoid contact with the sides of the tube. 5. Keeping the loop hand still, do aseptic technique with the tube then change the cotton plug. 6. Inoculate on plated, tubed or broth media. 7. Flame the inoculating loop red hot after its use.
SLANT-TUBE MEDIA 1. Heat the inoculating loop/needle red hot. 2. Remove and hold the cotton plug with the little finger where your loop hand is. 3. Do aseptic technique by passing the lid of the tube through the flame. 4. With the agar surface facing upward, hold the open tube at an angle. 5. Insert the loop/needle until its tip is over the desired position. Gently touch the colonies and avoid touching the sides of the tube. 6. Keeping the loop hand still, do aseptic technique with the tube then change the cotton plug. 7. Inoculate on plated, tubed or broth media. 8. Flame the inoculating loop red hot after its use.