MacConkey Agar (MAC): Composition, preparation, uses and colony characteristicsAUGUST 14, 2013 BY TANKESHWAR ACHARYAIN CULTURE MEDIA USED IN MICROBIOLOGY, LABORATORY DIAGNOSIS OF BACTERIAL DISEASE, MICROBIOLOGY · 5 COMMENTS MacConkey agar was developed in 20th century by Alfred Theodore MacConkey. It was the first formulated solid differential media. MacConkey Agar is a selective and differential culture media commonly used for the isolation of enteric Gram-negative bacteria. It is based on the bile salt-neutral red-lactose agar of MacConkey. Crystal violet and bile salts in incorporated in MacConkey Agar to prevent the growth of gram-positive bacteria and fastidious gram-negative bacteria, such as Neisseria and Pasteurella. Gram-negative enteric bacteria can tolerate to bile salt because of their bile-resistant outer membrane. LF and NLF colonies in MacConkey Agar MacConkey Agar is selective for Gram negative organisms, and helps to differentiate lactose fermenting gram negative rods from Non lactose fermenting gram negative rods. It is primarily used for detection and isolation of members of family enterobacteriaceae and Pseudomonas spp. Composition of MacConeky Agar: 1. Enzymatic Digest of Gelatin, Casein and Animal tissue: provides nitrogen, vitamins, minerals and amino acids essential for growth. 2. Lactose: fermentable carbohydrate providing carbon and energy. 3. Bile Salts: selective agents and inhibit Gram positive organisms. 4. Crystal Violet: Use: Gram positive bacteria are generally inhibited by crystal violet. 5. Sodium Chloride: supplies essential electrolytes for transport and osmotic balance. 6. Neutral Red: pH indicator. which is red in color at pH’s below 6.8. When lactose is fermented, the pH of the medium decreases, changing the color of neutral red to pink the production of the acid drops the pH of the media. Intended use of MacConkey Agar: MacConkey Agar is used for the selective isolation and identification ofEnterobacteriaceae from feces. Strongly lactose fermenting bacteria produce sufficient acid which causes precipitation of the bile salts around the growth. Gram-negative bacteria that grow on MacConkey agar but do not ferment lactose appear colorless on the medium and the agar surrounding the bacteria remains relatively transparent. 3. urine. The drop in pH is indicated by the change of Neutral red indictor to pink (Neutral read appears pink at pH’s below 6. Pink halo in not seen around the colonies of weaker lactose fermenting bacteria. It appears as a pink halo surrounding individual colonies or areas of confluent growth. Non-lactose fermenting organisms grow as colorless or clear colonies Lactose Fermenter: . LINK: MacConkey Agar Plates (10/bx. You can purchase the ready made MacConkey Agar from the commercial suppliers. Suspend the measured amount of powder (See in the agar bottle and generally 50 gram) in 1 L of purified water and mix thoroughly. Expected Colony characteristics in MacConkey Agar Lactose-fermenting organisms grow as pink to brick red colonies with or without a zone of precipitated bile.8).7. wastewater and foods. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder. If the lactose is fermented by the bacteria. 2. Agar : Solidifying agent Note: Remember the ingredient used in bold letter Preparation of MacConkey Agar 1. Autoclave at 121°C for 15 minutes.) Principle behind Differential capability of MacConkey Agar Gram-negative enteric bacteria that grow on MacConkey agar are differentiated by their ability to ferment lactose. Shigella spp. S.: Mucoid lactose fermenter (MLF) 3. Other organisms showing colorless colonies are. Salmonella spp. therefore Non Lactose Fermenter (NLF) after 24 hours. 3. Hafnia spp. Shigella sonnei produces flat colonies with jagged edges. marcescens may be red pigmented. 4.. Providencia spp. colonies are light pink after 48 hours.: Late Lactose fermenter. Klebsiella spp.: Late Lactose fermenter.: NLF. Lactose fermenter (LF) after 48 hours. may be colorless to peach. may swarm depending on the amount of agar in the medium. characteristic foul smell 2. Flat. Yersinia spp.: NLF.: NLF 5. pink colonies with a surrounding darker pink area of precipitated bile salts.: NLF. Escherichia coli: Lactose fermenter. Serratia spp. No Growth: Gram positive bacteria Staphylococcus aureus: No growth . especially if the plate is left at 25oC Non Lactose Fermenter (NLF) 1. dry. Edwardsiellaspp. Proteus spp. Morganella spp. 4..Mixed growth of mucoid Lactose fermenting colonies and NLF colonies in MacConkey Agar 1. 2. Citrobacter spp. As a sterility test. Do you remember what is Mannitol? It’s a sugar. aureus from the clinical specimen. Grow an E. vitamin and carbon) 2. In this post I am discussing about MSA.5%): Salt is the common ingredient but see the percentage. 3. 2. After 48 hours. but don’t remember why and when. vitamin and carbon) . its composition. Composition of Mannitol Salt Agar 1. 3. incubate an uninoculated plate for 48 hours at 35-37°C with ~5% CO2 (or in a candle-jar). Agar: the medium contains agar (a solidifying agent). uses and colony characteristics AUGUST 13. the sterility test plate should remain clear. LABORATORY DIAGNOSIS OF BACTERIAL DISEASE · 5 COMMENTS Are you familiar with Mannitol salt agar (MSA)? Have you ever used Mannitol salt agar in your laboratory? If you have used it. Mannitol Salt Agar (MSA) contains: 1. differential and indicator medium which is used to isolate and identify S. 2. coli Quality Control strain for 18-24 hours on a MacConkey Agar at 35-37°C with ~5% CO2 (or in a candle-jar). coli should appear as pink to red colonies. Enzymatic Digest of Casein (Source of nitrogen. Passing result: E. 2013 BY TANKESHWAR ACHARYAIN CULTURE MEDIA USED IN MICROBIOLOGY. Observe the MacConkey for specific colony morphology. Enzymatic Digest of Animal Tissue (Source of nitrogen. which is very high compared with other media. Mannitol (1%): Mannitol is one of the major ingredients.QUALITY CONTROL 1. Mannitol Salt Agar (MSA): Composition. Salt (7. Remember this statement: Mannitol Salt Agar (MSA) is a selective. uses and the colony of characteristics of organisms that grows on MSA. than you are in right place. 5% salt environment (growth indicates tolerance for high salt environment – no growth means intolerance). vitamin and carbon) 4. we try to find does a particular microorganism is able to ferment sugar present in this media or not? . MSA helps to demonstrate the ability of a bacterium to grow in a 7.2 at 25oC (Remember the ingredients in Bold letters) You can purchase prepared Mannitol Salt Agar from the commercial suppliers or get the Mannitol Salt Agar Powder and prepare the media in your laboratory.3. aureus in Mannitol Salt Agar (MSA). Beef Extract (Source of nitrogen. Sodium Chloride (Why?? see below) 6. So what’s the purpose of using salt in Mannitol Salt Agar? Incorporation of 7. Phenol Red (Indicator) 7.5% sodium chloride in the medium helps to select only those bacteria which can tolerate high salt concentrations. D-Mannitol : Only Carbohydrate source present in the medium 5. Species of staphylococci are able to tolerate this salt concentration but other pathogenic bacteria may not. Selective media for Staphylococcus spp Yellow colonies of S. Image source: ASM Why Mannitol (Sugar) is used in Mannitol Salt Agar? Whenever there is the use of any sugar in any media used in Microbiology.4 ~+mn~ 0. This concentration inhibits the growth of most other gram-positive and gram-negative bacteria Thus MSA selectively isolates Staphylococcus spp i.e. Agar Final pH: 7. may have yellow halo around colonies. MSA is also a differential medium. Staphylococcus aureus: Yellow colonies. roseus (red) produces pink colonies on MSA. in MacConkey Agar we try to find.e. we try to identify the isolate on the basis of sucrose fermentation. and oropharynx). M. Enterococcus faecalis and Enterococcus faecium (most common enterococcal species that has been isolated from human infections) are salt tolerant bacteria. producing yellow colored colonies on MSA. They can ferment mannitol and produce lactic acid. so the color of the media around the bacterial colony does not change to yellow. Staphylococcus epidermidis: Colorless to pink colonies 3. next task of microbiology laboratory (microbiologist) is differentiating S. . we try to find does a particular organism ferments Mannitol or not? You must be aware by now. TSI Agar. luteus (yellow) can produce yellow colonies.For example. Pathogenic staphylococci. while above pH 8. such as M. all the staphylococci spp are not pathogenic to human. aureus. the medium is a yellow color.9 to 8.4. Vibrio cholerae is able to ferment Sucrose and gives yellow color in TCBS. at pH levels below 6. But if Coagulase negative staphylococcus (CONS) grow. mucosa. Urea base agar andXLD Agar.4) the color of phenol red is red. the color of phenol red is pink. Troubleshooting: 1. they cant ferment Mannitol. Expected colony characteristics of organism in Mannitol Salt Agar 1. if that particular specimen contains S. So. Note: Other commonly used media that contain Phenol red as pH indicator are. Catalase test can help to differentiate between Enterococcus (-ve) and Staphylococcus (+ve). i. When grown on mannitol salt agar some species of Micrococcus (Micrococcus is a normal flora of human skin. it ferments Mannitol (whenever sugar is fermented acid is produced) and changes the pH of medium to acidic. So. producing yellow halo around colonies in MSA thus resembling with S. So. does a gram negative rod is lactose fermenter or not? In TCBS. aureus from other Staphylococcus spp. it appear pink. # Note: Staphylococcus saprophyticus (coagulse negative Staphylococcus) may ferment mannitol. Escherichia coli: Does not grow 2. Find out difference between Micrococcus and Staphylococcus here 2. As MSA contains phenol red as a pH indicator. Remember that in the neutral pH (6. aureus. Staphylococcus aureus is able to ferment Mannitol (It is coagulase test positive) but others (coagulase negative Staphylococcus) are not. Similarly in MSA.9. that otherwise wouldn’t grow. Composition of Blood Agar: Pancreatic digest of casein Papaic digest of soy meal NaCl Agar Distilled water Combine the ingredients and adjust the pH to 7. such as streptococci. Procedure for the preparation of Blood Agar . bacterial growth medium. Uses and Types of Hemolysis AUGUST 22. soybean protein digest. Heating of blood agar converts it into chocolate agar (heated blood turns a chocolate color) and supports the growth of these bacteria. such as streptococci. Tryptones). and sterilize by autoclaving. do not grow well on ordinary growth media.Blood Agar: Composition. MICROBIOLOGY · 17 COMMENTS Blood agar is an enriched. Boil to dissolve the agar. Blood agar consists of a base containing a protein source (e.. V factor) . agar and 5% sheep blood. Preparation.g.g. Blood agar is a type of growth medium (trypticase soya agar enriched with 5% sheep blood) that encourages the growth of bacteria. Blood contains inhibitors for certain bacteria such as Neisseria and Haemophilus genera and the blood agar must be heated to inactivate these inhibitors and to release essential growth factors (e. LABORATORY DIAGNOSIS OF BACTERIAL DISEASE. Fastidious organisms.3. sodium chloride (Nacl). Beta hemolysis in sheep blood agar. 2013 BY TANKESHWAR ACHARYAIN CULTURE MEDIA USED IN MICROBIOLOGY. The shelf life of thus prepared blood agar is up to four weeks. 3. The pH of the blood agar range from 7. Transfer thus prepared blood agar base to a 50°C water bath. pneumoniae: Alpha-hemolysis 3. (Note: If you are planning to prepare a batch of blood agar plates. 4.6 at room temperature. Prepare the blood agar base as instructed by the manufacturer. prepare few blood agar plates first to ensure that blood is sterile).1. Check for the growth characteristics of each species 1. Store the plates at 2-8°C. Inoculate also a plate withH. preferably in sealed plastic bags to prevent loss of moisture. When the agar base is cooled to 50°C. pyogenes: Beta-hemolysis 2. Dispense 15 ml amounts to sterile petri plates aseptically 5. influenzae . add sterile blood agar aseptically and mix well gently. S. 2.e. aureus (i. Incubate the plates in a carbon dioxide enriched atmosphere at 35-37°C overnight. Quality control of Blood Agar 1. 6. Satellitism Test). 4. influenzae and streak with S. S. pneumoniae.2 to 7. You must have warmed the blood to room temperature at the time of dispensing to molten agar base. Satellitism of H. 3. Label the medium with the date of preparation and give it a batch number (if necessary). Inoculate the plates with 5 hour broth cultures of Streptococcus pyogenes and S. Sterilize by autoclaving at 121°C for 15 minutes. Avoid formation of air bubbles. 2. How does one know if the colonies they are observing on a plate have caused alpha hemolysis or beta hemolysis? Note: To know the type of blood agar hemolysis. Determine the type of hemolysis. gamma hemolysis and alpha prime or wide zone alpha hemolysis. 2. Hemolysis is best observed by examining colonies grown under anaerobic conditions or inspecting subsurface colonies. the blood agar plate must be held up to a light source and observed with the light coming from behind (transmitted light). These hemolysin (extotoxin) radially diffuses outwards from the colony (or colonies) causing complete or partial destruction of the red cells (RBC) in the medium and complete denaturation of hemoglobin within the cells to colorless products. Beta hemolysis. Alpha hemolysis. Four types of hemolysis are produced in Sheep blood agar by Streptococci namely. identification (with the use of either Optochin disc orBacitracin disc and testing the sensitivity of the isolate) and antimicrobial susceptibility of Streptococci. .Optochin and Bacitracin Sensitivity of the isolates in Blood Agar Uses of Blood Agar: Blood agar has two major uses: 1. if any. Isolation. Blood Agar and Hemolysis Certain bacterial species produce extracellular enzymes that lyse red blood cells in the blood agar (hemolysis). Gamma or non hemolysis: No hemolysis of RBC. 3. beta hemolytic streptococci-Streptococcus agalactiace. But Streptococcus pneumoniae which is also alpha hemolytic causes serious pneumonia and other deadly infectious disease. Many of the alpha hemolytic streptococci are part of the normal body flora. causing a clearing of blood from the medium under and surrounding the colonies e. Beta Hemolysis: Complete lysis of Red Blood Cells. Oxygen labile SLO and oxygen stable SLS hemolysins is observed only in anaerobic conditions. then one will observe a zone of hemolysis surrounding a growing colony. α hemolysis is due to the reduction of RBC hemoglobin to methemoglobin in the medium surrounding the colony. . Group A beta hemolytic streptococci-Streptococcus pyogenes and Group B. No change of the medium under and surrounding the colonies. with a zone of complete red-cell hemolysis surrounding the zone of intact erythrocytes.If either type of hemolysis is present. Alpha hemolysis: Partial lysis of the RBC to produce a greenish-gray or brownish discoloration around the colony. For group A streptococci maximal activity of both the hemolysins. 4. Alpha prime or wide zone alpha hemolysis: A small zone of intact erythrocytes immediately adjacent to bacterial colony.g. Various types of Hemolysis 2. This type of hemolysis may be confused with Beta hemolysis. 1. influenzae. LABORATORY DIAGNOSIS OF BACTERIAL DISEASE. . when incubated at 35-37°C in a 5% CO2 atmosphere. CULTURE MEDIA USED IN MICROBIOLOGY. Target Hemolysis: Clostridium perfringens is readily identified in the laboratory by its characteristic “double zone” hemolysis also known has target hemolysis. uses and colony characteristics SEPTEMBER 8. It is used for the isolation of fastidious organisms. Chocolate Agar: Composition. 2013 BY TANKESHWAR ACHARYAIN BACTERIOLOGY. MICROBIOLOGY · 1 COMMENT Chocolate Agar (CAP) is the lysed blood agar.Double Zone hemolysis produced by Clostridium perfringens 5. The name is itself derived from the fact that red blood cell (RBC) lysis gives the medium a chocolate-brown color. the red blood cells are lysed. The composition of Chocolate agar and the Blood Agar is same and the only difference is while preparing Chocolate agar. such asHaemophilus. 2. meningitidis. 4. Passing result: . Neither of these species is able to grow on Sheep Blood Agar. incubate an uninoculated plate for 48 hours at 35-37°C with ~5% CO2 (or in a candle-jar). Neisseria meningitidis and Haemophilus spp. The most common species that require this enriched medium for growth include: Neisseria gonorrhoeae. Dispense 20 ml into 15×100 mm Petri dishes. Grow N. Quality Control: 1. Place the plates in sterile plastic bags and store at 4ºC until use. Observe the CAP for specific colony morphology and hemolysis. Preparation of Chocolate Agar: 1. S. pneumoniae. Heat-lyse a volume of horse or sheep blood that is 5% of the total volume of media being prepared very slowly to 56°C in a water bath. 2. Hemin (factor X) is available from non-hemolyzed as well as hemolyzed blood cells. influenzae QC strains for 18-24 hours on a CAP at 3537°C with ~5% CO2 (or in a candle-jar). and H. 3. As a sterility test.Haemophilus influenzae on chocolate agar The lysis of RBC during the heating process releases intracellular coenzyme Nicotinamide adenine dinucleotide (NAD or V Factor) in to the agar for utilization by fastidious bacteria (the heating process also inactivates growth inhibitors). Allow the media to solidify and condensation to dry. moist. N.5-1 mm in diameter. smooth. exhibit smooth consistency and defined margins. smooth. meningitidis and H. After 48 hours. convex. 2. opaque colonies on the CAP with no discoloration of the medium. round. and are typically 0. glistening colonies with a clearly defined edge. Haemophilus influenzae: Non hemolytic. convex. influenzae should appear as large. non-hemolytic. 3. Neisseria meningitidis: Growth on chocolate agar is grayish. Neisseria gonorrhoeae: Colonies on Chocolate agar are pinkish-brown and translucent. S. the sterility test plate should remain clear Colony characteristics in chocolate agar 1. pneumoniae should appear as small grey to green colonies with a zone of alphahemolysis (only slightly green) on the CAP. colorless-to grey. opaque cream-to-gray colonies (accompanying Sheep blood agar shows no growth) . round.
Report "Principles of MacConkey Agar, Mannitol Salt Agar, Blood Agar Plate and Chocolate Agar Plate"