MacConkey Agar (MAC): Composition, preparation, uses and colony characteristics
AUGUST 14, 2013 BY TANKESHWAR ACHARYAI ACHARYAIN CULTURE MEDIA USED IN MICROBIOLOGY,, LABORATORY DIAGNOSIS OF BACTERIAL DISEASE, MICROBIOLOGY 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. I t 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 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 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: 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
7. Agar : Solidifying agent Note: Remember the ingredient used in bold letter
Preparation of MacConkey Agar
1. Suspend the measured amount of powder (See in the agar bottle and generally 50 gram) in 1 L of purified water and mix thoroughly. 2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder. 3. Autoclave at 121°C for 15 minutes. You can purchase the ready made MacConkey Agar from the commercial suppliers. LINK: MacConkey Agar Plates (10/bx.) Principle behind Differential capability of MacConkey Agar
Gram-negative enteric bacteria that grow on MacConkey agar are differentiated by their ability to ferment lactose. If the lactose is fermented by the bacteria, the production of the acid dro ps the pH of the media. The drop in pH is indicated by the change of Neutral red indictor to pink (Neutral read appears pink at pH’s below 6.8).
Strongly lactose fermenting bacteria produce sufficient acid which causes precipitation of the bile salts around the growth. It appears as a pink halo surrounding individual colonies or areas of confluent growth. Pink halo in not seen around the colonies of weaker lactose fermenting bacteria. 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. Intended use of MacConkey Agar : MacConkey Agar is used for the selective isolation and identification
of Enterobacteriaceae from feces, urine, wastewater and foods. Expected Colony characteristics in MacConkey Agar
Lactose-fermenting organisms grow as pink to brick red colonies with or without a zone of precipitated bile. Non-lactose fermenting organisms grow as colorless or clear colonies Lactose Fermenter:
Mixed growth of mucoid Lactose fermenting colonies and NLF colonies in MacConkey Agar 1. Citrobacter spp.: Late Lactose fermenter; therefore Non Lactose Fermenter (NLF) after 24 hours; Lactose fermenter (LF) after 48 hours; colonies are light pink after 48 hours. 2. Klebsiella spp.: Mucoid lactose fermenter (MLF) 3. Escherichia coli : Lactose fermenter; Flat, dry, pink colonies with a surrounding darker pink area of precipitated bile salts. 4. Serratia spp.: Late Lactose fermenter; S. marcescens may be red pigmented, especially if the plate is left at 25oC Non Lactose Fermenter (NLF)
1. Proteus spp.: NLF; may swarm depending on the amount of agar in the medium; characteristic foul smell 2. Shigella spp.: NLF; Shigella sonnei produces flat colonies with jagged edges. 3. Yersinia spp.: NLF; may be colorless to peach. 4. Salmonella spp.: NLF 5. Other organisms showing colorless colonies are; Edwardsiellaspp, Hafnia spp., Morganella spp., Providencia spp. No Growth: Gram positive bacteria
Staphylococcus aureus: No growth
QUALITY CONTROL
1. Grow an E. coli Quality Control strain for 18-24 hours on a MacConkey Agar at 35-37 °C with ~5% CO2 (or in a candle-jar). 2. Observe the MacConkey for specific colony morphology. 3. As a sterility test, incubate an uninoculated plate for 48 hours at 35 -37°C with ~5% CO2 (or in a candle-jar). Passing result:
E. coli should appear as pink to red colonies.
After 48 hours, the sterility test plate should remain clear.
Mannitol Salt Agar (MSA): Composition, uses and colony characteristics
AUGUST 13, 2013 BY TANKESHWAR ACHARYAIN CULTURE MEDIA USED IN MICROBIOLOGY, LABORATORY DIAGNOSIS OF BACTERIAL DISEASE · 5 COMMENTS Are you familiar with Mannitol salt agar (MSA)? Have you ever used Mannitol salt ag ar in your laboratory? If you have used it, but don’t remember why and when, than you are in right place. In this
post I am discussing about MSA, its composition, uses and the colony of characteristics of o rganisms that grows on MSA. Remember this statement: Mannitol Salt Agar (MSA) is a selective, differential and indicator medium
which is used to isolate and identify S. aureus from the clinical specimen. Mannitol Salt Agar (MSA) contains: 1. Mannitol (1%): Mannitol is one of the major ingredients. Do you remember what is Mannitol? It’s a sugar.
2. Salt (7.5%): Salt is the common ingredient but see the pe rcentage, which is very high compared with other media. 3. Agar: the medium contains agar (a solidifying agent). Composition of Mannitol Salt Agar
1. Enzymatic Digest of Casein (Source of nitrogen, vitamin and carbon) 2. Enzymatic Digest of Animal Tissue (Source of nitrogen, vitamin and carbon)
3. Beef Extract (Source of nitrogen, vitamin and carbon) 4. D-Mannitol : Only Carbohydrate source present in the medium 5. Sodium Chloride (Why?? see below) 6. Phenol Red (Indicator) 7. Agar Final pH: 7.4 ~+mn~ 0.2 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. So what’s the purpose of using salt in Mannitol Salt Agar?
Incorporation of 7.5% sodium chloride in the medium helps to select only those bacteria which can tolerate high salt concentrations. MSA helps to demonstrate the ability of a ba cterium to grow in a 7.5% salt environment (growth indicates tolerance for high salt environment – no growth means intolerance). Species of staphylococci are able to tolerate t his salt concentration but other pathogenic bacteria may not. This concentration inhibits the growth of most other gram-positive and gram-negative bacteria Thus MSA selectively isolates Staphylococcus spp i.e. Selective media for Staphylococcus spp
Yellow colonies of S. aureus in Mannitol Salt Agar (MSA). 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, we try to find d oes a particular microorganism is able to ferment sugar present in this media or not?
For example, in MacConkey Agar we try to find, does a gram negative rod is lactose fermenter or not? In TCBS, we try to identify the isolate on the basis of sucrose fermentation. Vibrio cholerae is able to ferment Sucrose and gives yellow color in TCBS. Similarly in MSA, we try to find does a particular organism ferments Mannitol or not? You must be aware by now; all the staphylococci spp are not pathogenic to human. So, ne xt task of microbiology laboratory (microbiologist) is differentiating S. aureus from other Staphylococcus spp. Pathogenic staphylococci, i.e. Staphylococcus aureus is able to ferment Mannitol (It is coagulase test positive) but others (coagulase negative Staphylococcus) are not. So, if that particular specimen contains S. aureus, it ferments Mannitol (whenever sugar is fermented acid is produced ) and changes the pH of medium to acidic. As MSA contains phenol red as a pH indicator, at pH levels below 6.9, the medium is a yellow color. But if Coagulase negative staphylococcus (CONS) grow, they cant ferment
Mannitol, so the color of the media around the bacterial colony does not change to yellow, it appear pink. # Note: Staphylococcus saprophyticus (coagulse negative Staphylococcus) may ferment mannitol, producing yellow halo around colonies in MSA thus resembling with S. aureus. So, MSA is also a differential medium. Remember that in the neutral pH (6.9 to 8.4) the color of phenol red is red; while above pH 8.4, the color of phenol red is pink. Note: Other commonly used media that contain Phenol red as pH indicator are; TSI Agar , Urea base
agar and XLD Agar. Expected colony characteristics of organism in Mannitol Salt Agar
1. Escherichia coli : Does not grow 2. Staphylococcus epidermidis: Colorless to pink colonies 3. Staphylococcus aureus: Yellow colonies; may have yellow halo around colonies. Troubleshooting:
1. When grown on mannitol salt agar some species of Micrococcus (Micrococcus is a normal flora of human skin, mucosa, and oropharynx), such as M. luteus (yellow) can produce yellow colonies. M. roseus (red) produces pink colonies on MSA. Find out difference between Micrococcus and Staphylococcus here 2. Enterococcus faecalis and Enterococcus faecium (most common enterococcal species that has been isolated from human infections) are salt tolerant bacteria. They can ferment mannitol and
produce lactic acid, producing yellow colored colonies on MSA. Catalase test can help to differentiate between Enterococcus (-ve) and Staphylococcus (+ve).
Blood Agar: Composition, Preparation, Uses and Types of Hemolysis
AUGUST 22, 2013 BY TANKESHWAR ACHARYAIN CULTURE MEDIA USED IN MICROBIOLOGY, LABORATORY DIAGNOSIS OF BACTERIAL DISEASE, MICROBIOLOGY · 17 COMMENTS Blood agar is an enriched , bacterial growth medium . Fastidious organisms, such as streptococci, do not grow well on ordinary growth media. Blood agar is a type of growth medium (trypticase soya agar enriched with 5% sheep blood ) that encourages the growth of bacteria, such as streptococci, that otherwise wouldn’t grow.
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.g., V factor) . Heating of blood agar converts it into chocolate agar (heated blood turns a chocolate color) and supports the growth of these bacteria.
Beta hemolysis in sheep blood agar. Blood agar consists of a base containing a protein source (e.g. Tryptones), soybean protein digest, sodium chloride (Nacl), agar and 5% sheep blood. 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 7.3. Boil to diss olve the agar, and sterilize by autoclaving. Procedure for the preparation of Blood Agar
1. Prepare the blood agar base as instructed by the manufacturer. S terilize by autoclaving at 121°C for 15 minutes. 2. Transfer thus prepared blood agar base to a 50°C water bath. 3. When the agar base is cooled to 50°C, add sterile blood agar aseptically and mix well gently. Avoid formation of air bubbles. You must have warmed the blood to room temperature at the time of dispensing to molten agar base. (Note: If you are planning to prepare a batch of blood agar plates, prepare few blood agar plates first to ensure that blood is sterile). 4. Dispense 15 ml amounts to sterile petri plates aseptically 5. Label the medium with the date of preparation and give it a batch number (if necessary). 6. Store the plates at 2-8°C, preferably in sealed plastic bags to prevent loss of moisture. The shelf life of thus prepared blood agar is up to four weeks. Quality control of Blood Agar
1. The pH of the blood agar range from 7.2 to 7.6 at room temperature. 2. Inoculate the plates with 5 hour broth culture s of Streptococcus pyogenes and S. pneumoniae. Inoculate also a plate withH. influenzae and streak with S. aureus (i.e. Satellitism Test ).
3. Incubate the plates in a carbon dioxide enriched atmosphere at 35 -37°C overnight. 4. Check for the growth characteristics of each species 1. S. pyogenes: Beta-hemolysis 2. S. pneumoniae: Alpha-hemolysis 3. Satellitism of H. influenzae
Optochin and Bacitracin Sensitivity of the isolates in Blood Agar Uses of Blood Agar:
Blood agar has two major uses: 1. Isolation, identification (with the use of either Optochin disc orBacitracin disc and testing the sensitivity of the isolate) and antimicrobial susceptibility of Streptococci. 2. Determine the type of hemolysis, if any. Blood Agar and Hemolysis
Certain bacterial species produce extracellular enzymes that lyse red blood cells in the blood agar (hemolysis). These hemolysin (extotoxin) radially diffuses outwards from the colony (or colonies) causing complete or partial destruction of the red cells (RB C) in the medium and complete denaturation of hemoglobin within the cells to colorless products. Four types of hemolysis are produced in Sheep blood agar by Streptococci namely; Alpha hemolysis, Beta hemolysis, gamma hemolysis and alpha prime or wide zone alpha hemolysis. Hemolysis is best observed by examining colonies grown under anaerobic conditions or inspecting subsurface colonies. 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, the blood agar plate must be held up to a light source
and observed with the light coming from behind (transmitted light).
If either type of hemolysis is present, then one will observe a zone of hemolysis surrounding a growing colony. 1. Alpha hemolysis: Partial lysis of the RBC to produce a greenish -gray or brownish discoloration around the colony. α hemolysis is due to the reduction of RBC hemoglobin to methemoglobin in
the medium surrounding the colony. Many of the alpha hemolytic streptococci are part of the normal body flora. But Streptococcus pneumoniae which is also alpha hemolytic causes serious pneumonia and other deadly infectious disease.
Various types of Hemolysis 2. Beta Hemolysis: Complete lysis of Red Blood Cells, causing a clearing of blood from the medium under and surrounding the colonies e.g. Group A beta hemolytic streptococci- Streptococcus pyogenes and Group B, beta hemolytic streptococci-Streptococcus agalactiace. For group
A streptococci maximal activity of both the hemolysins; Oxygen labile SLO and oxygen stable SLS hemolysins is observed only in anaerobic conditions. 3. Gamma or non hemolysis: No hemolysis of RBC. No change of the medium under and surrounding the colonies. 4. Alpha prime or wide zone alpha hemolysis : A small zone of intact erythrocytes immediately adjacent to bacterial colony, with a zone of c omplete red-cell hemolysis surrounding the zone of intact erythrocytes. This type of hemolysis may be confused with Beta hemolysis.
Double Zone hemolysis produced by Clostridium perfringens 5. Target Hemolysis: Clostridium perfringens is readily identified in the laboratory by its characteristic “double zone” hemolysis also known has target hemolysis.
Chocolate Agar: Composition, uses and colony characteristics
SEPTEMBER 8, 2013 BY TANKESHWAR ACHARYAIN BACTERIOLOGY, CULTURE MEDIA USED IN MICROBIOLOGY, LABORATORY DIAGNOSIS OF BACTERIAL DISEASE, MICROBIOLOGY · 1 COMMENT Chocolate Agar (CAP) is the lysed blood agar. The name is itself derived from the fact that red blood cell (RBC) lysis gives the medium a chocolate-brown color. It is used for the isolation of fastidious organisms, such asHaemophilus. influenzae , when incubated at 35-37°C in a 5% CO2 atmosphere. The composition of Chocolate agar and the Blood Agar is same and the only difference is while preparing Chocolate agar, the red blood cells are lysed.
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). Hemin (factor X) is available from non-hemolyzed as well as hemolyzed blood cells. The most common species that require this enriched medium for growth include: Neisseria gonorrhoeae, Neisseria meningitidis and Haemophilus spp. Neither of these species is able to gro w on
Sheep Blood Agar. Preparation of Chocolate Agar:
1. 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. Dispense 20 ml into 15×100 mm Petri dishes. Allow the media to solidify and condensation to dry. 3. Place the plates in sterile plastic bags and store at 4º C until use. 4. As a sterility test, incubate an uninoculated plate for 48 hours at 35 -37°C with ~5% CO2 (or in a candle-jar). Quality Control :
1. Grow N. meningitidis, S. pneumoniae, and H. influenzae QC strains for 18-24 hours on a CAP at 3537°C with ~5% CO2 (or in a c andle-jar). 2. Observe the CAP for specific colony morphology and hemolysis. Passing result:
N. meningitidis and H. influenzae should appear as large, round, smooth, convex, colorless-to
grey, opaque colonies on the CAP with no discoloration of the medium.
S. pneumoniae should appear as small grey to green colonies with a zone of alpha-
hemolysis (only slightly green) on the CAP.
After 48 hours, the sterility test plate should remain clear
Colony characteristics in chocolate agar
1. Neisseria meningitidis : Growth on chocolate agar is g rayish, non-hemolytic, round, convex, smooth, moist, glistening colonies with a clearly defined edge. 2. Neisseria gonorrhoeae : Colonies on Chocolate agar are pinkish-brown and translucent, exhibit smooth consistency and defined margins, and are typically 0.5-1 mm in diameter. 3. Haemophilus influenzae : Non hemolytic, opaque cream-to-gray colonies (accompanying Sheep blood agar shows no growth)
