MacConkey's Agar is a culture medium designed to grow up Gram-negative bacteria and stain them for lactose fermentation.

It contains bile salts, crystal violet dye (to inhibit Gram-positive bacteria), neutral red dye (which stains microbes fermenting lactose), lactose and peptone. Alfred Theodore MacConkey developed it while working as a bacteriologist for the Royal Commission on Sewage Disposal.

A selective medium is a substance (usually agar-based) which grows a specific type of microbe. Using different nutrient ratios, chemical compounds (e.g. methylene blue) and incubation times, speciation of bacteria can be performed before to use other methods for the presuntive identification of bacteria or fungus.
Examples Eosin-methylen blue agar (EMB) that contains methylene blue which is toxic for Gram-positive bacteria allowing only the growth (thus selecting) of Gram negative bacteria. YM (Yeast and Mold) which has a low pH, deterring bacterial growth. Blood agar (used in strep tests), which contains beef heart blood which becomes transparent in the presence of hemolytic Streptococcus. MacConkey's Agar for Gram-negative bacteria.

Selective genes These bacterial transgenes interact with selective growth media: GAL transgene: o + growth on galactose o - no growth on glucose URA3 transgene: o + growth on uracil-deficient medium o - no growth on FOA medium

An agar plate is a sterile Petri dish that contains agar plus nutrients, and is used to culture microorganisms. Generally, selecting substances are also added to the plate, such as antibiotics.
Preparation of agar plates Most types of agar are purchased pre-prepared in powder form, although it is possible to buy a base agar mix and add nutrients separately. They are dissolved in distilled water as per their instructions. It is usually necessary to gently boil the mixture to facilitate dissolving: this can be done in a microwave oven, or over a gentle flame. Once dissolved the agar needs to be sterilised, usually by pouring it into a Erlenmeyer flask, then sealing the top with a cotton wool wad, and finally covering the cotton wool with a loose layer of aluminium foil. This is then autoclaved for 15 minutes. The sterile agar is then allowed to cool to 50 C: this is just above the setting point of agar and pouring at this cooler temperature helps prevent condensation forming on the lid. Chemical agents (e.g. antibiotics) are added at this step to prevent their degradation at the higher temperatures; agar is especially useful in that it may be sterilized at higher temperatures yet since it solidifies at lower temperatures, chemical additives may be added without fear of degradation. Before the plates are poured, every care is taken not to contaminate them with stray bacteria: sterile technique must be used.

1. Hands are thoroughly washed with antimicrobial soap and hot water.

2. The bench is wiped with ethanol or some other disinfectant 3. A Bunsen burner is set up with a gentle blue flame. This will be used to sterilise the mouth of the
4. 5. 6. 7. 8. 9. flask, and will also provides a reasonably sterile environment in the vicinity. This will also be used to flame plates that develop bubbles from pouring. The number of plates to be poured are placed on the bench, with their lids still on. The aluminium foil is removed and discarded. The cotton wool is removed with the little finger. It is held in the little finger the whole time, not put down. The mouth of the flask is flamed to kill bacteria on the outside of the rim. The lid of the plate is lifted just high enough to allow the plate to be poured, and the dish is quickly half filled with agar. The lid is replaced, the plate swirled gently to ensure even distribution of the molten agar, then left to stand on the bench for at least 20 minutes to solidify. Once all the plates are poured, the flask mouth is reflamed and the cotton wool reinserted. Any unused agar is still sterile.

10. To allow traceability, a lot number assigned to the flask is written on the plates poured with it. [edit] Inoculation techniques Before inoculation, important information is written on the bottom of the plates, close to the rim: 1. 2. 3. 4. date of inoculation temperature of incubation duration of incubation microorganism inoculated

Streaking The most common method of inoculating an agar plate is streaking.

Streak plates

1. With this method, a small amount of sample is placed on the side of the agar plate (either with a
2. 3. 4. swab, or as a drop from an inoculating loop if the sample is a liquid). A sterile loop (flamed until red hot, then cooled by touching the agar away from the inoculated sample) is then used to spread the bacteria out in one direction from the initial site of inoculation. This is done by moving the loop from side to side, passing through the initial site. The loop is then sterilised (by flaming) again and the first streaks are then spread out themselves. This is repeated 2-3 times, moving around the agar plate.

What should happen is that single bacterial cells get isolated by the streaking, and when the plate is incubated, forming discrete colonies that will have started from just one bacterium each.
Christmas tree

This pattern is used for culture of urine. A small loop is dipped in the urine, and a single streak is made down the middle of the agar plate. Then the loop is swayed in and out going through the streak multiple times at right-angles to the first streak.
Stab culture

A needle is flamed then immersed in the culture. It is then stabbed into a small sterile jar of nutrient agar. If the bacteria are anaerobic they will grow, otherwise they do not.
Preparing a lawn A lawn is often used for testing sensitivity to antibiotics, or for work with bacteriophages. What is needed is an even and complete spread of growth all over the agar plate (a "lawn"). Around an antibiotic disc there will be a clear area in which bacterial growth is inhibited; the diameter of this area can be measured to find out whether that bacterial strain is resistant to the antibiotic. One way to prepare a lawn is to use a 0.5 McFarland suspension of bacteria in saline (this means the saline is made just slightly turbid.) A sterile swab is dipped into this suspension, then it is moved from side to side down the whole agar plate so all the area is covered. The plate is rotated 90 and the swab moved side to side perpedicularly to the first time. This is done once more with the swab rotated 45 . Once a lawn has been prepared, a small disk of sterile filter paper is soaked in antibiotic and placed on the plate. After incubation there will be a ring of zero growth visible around the filter paper if the lawn bacteria are sensitive to that antibiotic. A collection of small disks each soaked in a different antibiotic, and attached to a larger ring, can be purchased commercially. They are known as antibiotic sensitivity discs or by the name "Mastrings" (a trademark of Mast Group Ltd) and can be used to test the sensitivity of an organism to a range of antibiotics all at once. Incubation of agar plates Plates are incubated upside down to prevent drops of condensation from collecting on the inoculated surface. Most plates are incubated at 37 C in a 5% CO2 atmosphere: the temperature and conditions that most of the body's bacteria will grow. Special incubators can maintain these conditions. Some bacteria must be incubated anaerobically (without any oxygen). These can be placed in containers, along with a substance that removes oxygen, and the tightly sealed container placed in the regular incubator. Fungi, and some bacteria (e.g. Yersinia sp.), should be incubated slightly cooler. This is usually 30 C, and room air often is used. Yoghurt bacteria grow at much higher temperatures (typically ~45 C). They are therefore particulally safe to use when teaching microbiology, especially to children. Campylobacter is a difficult bacterial species to grow. It needs special agar plates, plus its own microaerophilic environment.

Types of agar plates

Blood agar - contains blood cells from an animal (e.g. a sheep). It is an enriched differential media used to isolate fastidious organisms and detect hemolytic activity. -hemolytic activity will show complete lysis of red blood cells surrounding colony, while -hemolysis will only partially lyse hemoglobin and will appear green. -hemolysis is the term referring to a lack of hemolytic activity. Chocolate agar - this contains lysed blood cells, and is used for growing fastidious (fussy) respiratory bacteria. Sabouraud agar - used for fungi. It contains gentamicin and has a low pH that will kill most bacteria. Thayer-Martin agar - chocolate agar designed to isolate Neisseria gonorrhoeae. Hay Infusion agar - used for the culturing of slime moulds. MacConkey agar - is a selective differential media used to differentiate between Gram negative bacteria while inhibiting the growth of Gram positive bacteria. The addition of bile salts and crystal violet to the agar will inhibit the growth of most Gram positive bacteria, making MacConkey agar selective. Lactose and neutral red are added to differentiate the lactose fermenters, which form pink colonies, from lactose nonfermenters that form clear colonies. Mannitol Salt Agar (MSA) - is a selective differential media. Mannitol is the differential part, it indicates organisms that ferment mannitol. If mannitol fermentation is occurring, lactic acid will be produced, and the pH will drop causing the MSA plate to turn yellow. The salt portion is selective for halophiles; organisms that cannot withstand a high salt content will be unable to grow. Neomycin agar - contains the antibiotic neomycin. Nutrient agar - safe to use in school science laboratories because it does not selectively grow pathogenic bacteria. nz agar - The most important advantage of this culture medium is that it allows more rapid bacteriological diagnosis as, Salmonella and Shigella colonies can be clearly and reliably differentiated from other Enterobacteriaceae. The yields of Salmonella from stool samples obtained, when using this medium, are higher than those obtained with LEIFSON Agar or Salmonella Shigella Agar. XLD agar - xylose lysine deoxycholate agar. It is used for the culture of stool samples, and contains two indicators. It is formulated to inhibit Gram-positive bacteria, while the growth of Gram-negative bacilli is encouraged. The colonies of lactose fermenters appear yellow.

Safe disposal of agar plates Plates, once finished with, must be made safe before throwing away. The usual method is to place inside an autoclave bag and then sterilise by autoclaving at 121 C, 103 kPa (15 psi) for 15 minutes. Plastic plates will melt (hence the bag). After about 20 minutes the autoclave will have cooled down and the bag can safely be thrown away. If no autoclave is available an ordinary domestic pressure cooker can be used, or, in a hospital or professional lab an incinerator may be used. Other equipment may be decontaminated by being placed in a suitable disinfectant such as Virkon for 24 hours. When all manipulations are done, the bench is disinfected once again. The last step should be to wash the hands thoroughly with antimicrobial soap and hot water before leaving the lab.

Agar is a galactose polymer (or agarose) obtained from the cell walls of some species of red algae or seaweed (Sphaerococcus euchema) and species of Gelidium and Gracilaria, chiefly from eastern Asia, Chile and California. It is also known as Kanten, Agar-Agar, or Agal-Agal (Ceylon Agar). Chemically, agar is a polymer made up of subunits of the sugar galactose; it is a component of the algae's cell walls. Dissolved in hot water and cooled, agar becomes gelatinous; its chief use is as a culture medium for microbiological work. Other uses are as a laxative, a vegetarian gelatin substitute a thickener for soups, in jellies, ice cream and Japanese desserts such as anmitsu, as a clarifying agent in brewing, and for paper sizing fabrics. Uses in cooking Agar-agar is typically sold as packaged strips of washed and dried seaweed, or in powdered form. Raw agar is white and semi-translucent. For making jelly, it is boiled in water at a concentration of about 0.7-1% w/v (e.g. a 7 gram packet of powder into 1 litre of water would be 0.7%) until the solids dissolve, after which sweeteners, flavouring, colouring, and pieces of fruit may be added. The agar-agar may then be poured into molds or incorporated into other desserts, such as a jelly layer on a cake. Uses in microbiology Nutrient agar is used throughout the world as a medium for the growth of bacteria and fungi, but not viruses. Though less than 1% of all existing bacteria can be grown successfully, the basic agar formula can be used to grow most of the microbes, whose needs are known. More specific nutrient agars are available, because microbes can be picky. For example, blood agar, which is generally combined with horse blood, can be used to detect the presence of haemorrhagic micro-organisms such as E.coli O:157 H:7. The bacteria digest the blood, turning the plate clear. Agarose is also used in Agarose gel electrophoresis. Selective Media Selective media is agar specially treated to apply a selective pressure to organisms growing on it -- for example, to select for salt-tolerant, gram-positive, or gram-negative bacteria. Differential Media Differential media includes an indicator that causes visible, easily detectable changes in the appearance of the agar gel or bacterial colonies in a specific group of bacteria. For example, EMB (Eosin Methylene Blue) agar causes E. Coli colonies to have a metallic green sheen, and MSA (Mannitol Salt Agar) turns yellow in the presence of mannitol fermenting bacteria. Hysteresis Hysteresis describes the phenomenon of the differing liquid-solid state transition temperatures that agar exhibits. Agar melts at 85 C and solidifies from 32-40 C. A growth medium is an object in which microorganisms or cells in experience growth. There are different sorts of media for growing different sorts of cells. The biggest difference in growth media are between those used for cell culture uses specific cell types derived from plants or animals, and microbiological culture used for growing microorganisms, usually bacteria or yeast. These differences arise due to the fact that cells derived from whole organisms and grown in culture are often incapable of growth without the provision of certain requirements, such as hormones or growth factors which usually occur in vivo. In the case of animal cells these requirements are often provided by the addition of blood serum to the medium. These media are often red or pink due to the inclusion of pH indicators. In the case of microorganisms, there are no such limitations as they are often unicellular organisms. One other major difference is that

animal cells in culture are often grown on a flat surface to which they attach, and the medium is provided in a liquid form, which covers the cells. Bacteria such as Escherichia coli may be grown on solid media or in liquid media, liquid nutrient medium is commonly called nutrient broth. The most common growth media for microorganisms are nutrient broth or Luria-Bertani medium (L-B medium). Bacteria grown in liquid cultures often form colloidal suspensions. When agar, a substance which sets into a gel, is added to a liquid medium it can be poured into petri dishes where it will solidify, called agar plates, and provide a solid medium on which microbes may be cultured. Another important distinction between different growth media is that of a defined and an undefined medium. A defined medium will have known quantities of all ingredients, for microorganisms they consist of providing trace elements, any vitamins required by the microbe and especially a defined carbon source and nitrogen source (for example glucose or glycerol are often used as carbon sources, ammonium salts or nitrates as inorganic nitrogen sources), minimal media are defined media. Nutrient media are not defined media as they contain ingredients such as yeast extract which vary in composition depending on the source. This definition applies to cell culture media as well, where any medium containing, for example, animal blood serum is undefined, as the composition of the serum will vary from supplier to supplier and batch to batch. A good example of a growth medium is the wort used to make beer. The wort contains all the nutrients required for yeast growth, and under anaerobic conditions alcohol is produced. When the fermentation process is complete the microbe is removed from the medium which is now beer and ready for consumption. Some organisms require specialized environments, for example, since viruses are obligatory intracellular parasites, they require a growth medium composed of living cells. Often special media are required for microorganism and cell culture growth. Selective media are used to grow cells which possess a desired selectable trait. For example if a microorganism is resistant to a certain antibiotic, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing. Differential media are used to distinguish one microorganism type from another growing on the same media. While selective media are used to allow the growth of only select microoganisms, differential media allow the growth of multiple types, but result in distinguishing characteristics (such as the production of a brightly coloured or phosphorescent dye). Growth media can be both selective and differential. Principle: Microbiological method Mode of Action This culture medium represents a rich nutrient base, which provides optimal growth conditions for all relevant microorganisms. The pH value of 6.8 stabilizes the red blood corpuscles and favours the formation of clear haemolysis zones (NORTON 1932). Fresh, defibrinated sheep blood is most suitable for determining haemolysis forms. Boiled blood agar ("chocolate agar") is an extremely rich culture medium and can be prepared by heating after the blood has been added. If the culture medium base is to be used without blood, the pH should, however, be adjusted to 7.2 to 7.4 since most bacterial colonies appear somewhat earlier and grow better in a slightly alkaline medium. TARSHIS and FRISH (1951) recommended addition of 1 % glycerol and 25 % human blood when isolating tubercle bacilli from sputum, since recognizable mycobacteria colonies grow from even minimal amounts of sample material. HOSTY et al. (1953) reported, however, that 0.1 % glycerol and 2.5 % human blood together with 100 IU/mol of penicillin as a selective agent are sufficient. According to SONDAG et al. (1977) and BLACK a. VAN BUSKIRK (1973), addition of 5 mg/l gentamicin (e.g. 0.1 ml gentamicin solution) to blood agar permits selective cultivation of Streptococcus pneumoniae and other Streptococci as well as bacterioides, Clostridium and yeasts. For the selective cultivation of Aeromonas MISHRA et al. (1987) recommend an ampicillin sheep blood agar (ASBA 30).

Typical Composition (g/litre) Nutrient substrate (heart extract and peptones) 20.0; sodium chloride 5.0: agar-agar 15.0. Also to be added: Blood 50-80 ml. Preparation and storage Cat. No. 1.10886 Blood Agar Base (500 g) Usable up to expiry date when stored dry and tightly closed at +15 to +25 C. Protect from light. After first opening of the bottle the content can be used up to expiry date when stored dry and tightly closed at +15 to +25 C. Suspend 40 g/litre, autoclave (15 min at 121 C), cool to 45-50 C, add 5-8 % defibrinated blood, mix. pH: 6.8 0.2 at 25 C. Before adding blood, the prepared medium is clear and yellowish-brown, then blood coloured and not haemolytic. Poured blood plates can be stored for a maximum of 3 months in the refrigerator. preparation of boiled blood agar: after adding the blood, heat the culture medium for about 10 minutes at approx. 80 C with frequent swirling until it turns brownish (chocolate colour).

Experimental Procedure and Evaluation Inoculate the surface of the plates. Incubation: under optimal conditions usually 24 hours at 35 C aerobically (Cl. perfringens anaerobically). Check the plates for kind of hemolysis. Quality control Test strains

Inoculum (cfu/ml) Staphylococcus aureus ATCC 25923 103-105 Streptococcus pyogenes ATCC 103-105 12344 Streptococcus agalactiae ATCC 103-105 13813 Streptococcus pneumoniae ATCC 103-105 6301 Listeria monocytogenes ATCC > 105 19118 Bacillus cereus ATCC 11778 > 105 Clostridium perfringens ATCC 103-105 13124

Recovery rate (%) 70 70 70 70 70 70 70 (anaerobic incubation)

Hemolysis

Bacitracin test +

Additives Merck Cat.No. CN Biosciences

Product Ampicillin mono-sodium salt

Pack Size

1.01611.0001 1.04094.0500 CN Biosciences 1.07040.0001 1.11977.0001 1.13807.0001 1.13829.0001 1.14226.0001 1.15112.0001 1.16387.0001

Anaerocult A mini Glycerol (about 87 %) Penicillin G potassium salt Plate basket Gentamicin solution Anaerocult P Anaerocult A Anaeroclip Anaerotest Anaerobic jar Blood

1 x 25 500 ml 1ea 10 ml 1 x 25 1 x 10 1 x 25 1 x 50 1 ea

Staphylococcus aureus CHOCOLATE AGAR Chocolate agar is a nutrient medium which is used in culturing fastidious organisms such as Haemophilus species and Neisseria. It is comprised of sheep blood that provides the X and V factors necessary for Haemophilus growth. Chocolate agar, however, does not reveal hemolysis data, so species differentiation among the members of Haemophilus must be performed in another manner.

EOSIN METHYLENE BLUE (EMB) AGAR EMB agar is a differential medium used in identification and isolation of Gram-negative enteric rods. EMB agar also inhibits the growth of Gram-positive organisms. The differential basis of this medium involves two indicator dyes, eosin and methylene blue, that distinguish between lactose fermenting and non-lactose fermenting organisms. Lactose fermenters form colonies with dark centers and clear borders while the nonlactose fermenters form completely coloroless colonies.

MACCONKEY AGAR MacConkey agar is probably the most popular solid differential medium in the world. It is mainly used in identification of lactose fermenting, Gram-negative enteric pathogens and for inhibiting growth of Grampositive organisms. Bacterial colonies that can ferment lactose turn the medium red. This red color is due to the pH indicators response to the acidic environment created by fermenting lactose. Organisms that do not ferment lactose do not cause a color change.

BILE ESCULIN AGAR Bile esculin agar is a medium used to identify group D streptococci. This group of bacteria have the ability to grow in the presence of bile, an emulsifying agent produced in the liver. Group D streptococci also have the ability to hydrolyze esculin. This hydrolysis of esculin turns the medium black and denotes a positive test. Other bacteria capable of growing in the presence of bile do not turn the medium black. A variation of this medium uses sodium azide to inhibit the growth of all other Gram-positive bacteria and Gram-negative bacteria.