Mueller Hinton Agar (MHA)- Composition, Principle, Preparation, Results, Uses

Mueller Hinton Agar (MHA) is a type of microbiological growth medium that is widely used for antibiotic susceptibility testing of bacterial isolates. It was first developed in 1941 by John Howard Mueller and Jane Hinton, who were microbiologists working at Harvard University. They initially used this medium for the isolation of pathogenic Neisseria species, which are gram-negative bacteria that cause diseases such as gonorrhea and meningitis.

MHA is a nutrient-rich and non-selective medium, which means that it can support the growth of most non-fastidious bacteria, i.e., bacteria that do not require special nutrients or conditions to grow. It is also a non-differential medium, which means that it does not distinguish between different types of bacteria based on their appearance or biochemical reactions. MHA contains beef extract, acid hydrolysate of casein, starch, and agar as its main ingredients. Beef extract and acid hydrolysate of casein provides nitrogen, vitamins, carbon, amino acids, sulfur, and other essential nutrients for bacterial growth. Starch acts as a colloid and absorbs any toxic metabolites produced by bacteria, as well as mediates the rate of diffusion of antibiotics through the agar. Agar is the solidifying agent that gives the medium its gel-like consistency.

MHA is commonly used in the disk diffusion method, which is a simple and widely used method for testing the susceptibility of bacterial isolates to antibiotics. In this method, small disks impregnated with different antibiotics are placed on the surface of the agar, and the zone of inhibition around each disk is measured to determine the susceptibility of the bacterial isolate to that antibiotic. The zone of inhibition is the area where bacterial growth is prevented or reduced by the antibiotic. The size of the zone depends on several factors, such as the concentration and diffusion rate of the antibiotic, the type and number of bacteria, and the thickness and pH of the agar.

MHA has some advantages over other media for antibiotic susceptibility testing. It has a low content of calcium and magnesium ions, which can interfere with the activity of certain antibiotics, such as aminoglycosides, tetracyclines, and colistin. It also has a low content of thymidine and thymine, which are nucleotides that can inhibit the action of sulfonamides and trimethoprim, two antibiotics that target bacterial DNA synthesis. Moreover, MHA has good batch-to-batch reproducibility and quality control standards, which ensure consistent and reliable results.

MHA is also used for other purposes besides antibiotic susceptibility testing. It can be used to isolate and maintain Neisseria and Moraxella species, which are gram-negative bacteria that are part of the normal flora of the upper respiratory tract but can also cause infections such as otitis media and sinusitis. It can also be used for food testing and procedures commonly performed on aerobic and facultative anaerobic bacteria. Furthermore, MHA can be modified by adding sheep blood or hemoglobin to enhance the growth of Streptococcus pneumoniae and Haemophilus influenza, two gram-positive bacteria that cause respiratory infections such as pneumonia and bronchitis.

MHA is a versatile and useful medium for microbiology laboratories. It has been instrumental in the development of antibiotics and in the study of antibiotic resistance. However, it also has some limitations that need to be considered when using it for antibiotic susceptibility testing. For example, it may not support the growth of fastidious organisms or anaerobes, which require special nutrients or conditions to grow. It may also be affected by variations in pH or nutrient content that can alter the activity of certain antibiotics. Therefore, careful selection and preparation of MHA is important for accurate microbiological assays.

Mueller Hinton Agar (MHA) was originally designed for the isolation of pathogenic species of Neisseria, a genus of Gram-negative bacteria that includes the causative agents of gonorrhea and meningitis. Mueller and Hinton developed MHA in 1941 by modifying a medium that was previously used for cultivating gonococci. They found that MHA supported the growth of most strains of Neisseria and also allowed the differentiation of Neisseria species based on their carbohydrate fermentation patterns. MHA contains beef extract and acid hydrolysate of casein as sources of nutrients, starch as a colloid that absorbs toxic metabolites, and agar as a solidifying agent. The pH of MHA is adjusted to 7.3, which is optimal for the growth of Neisseria.

MHA is still used today for the isolation and identification of Neisseria species from clinical specimens such as blood, cerebrospinal fluid, genital swabs, and throat swabs. MHA can also be supplemented with blood, hemoglobin, or nicotinamide adenine dinucleotide (NAD) to enhance the growth of some fastidious Neisseria strains. MHA can also be used for performing biochemical tests such as oxidase, catalase, nitrate reduction, and carbohydrate fermentation on Neisseria isolates. MHA can also be modified by adding antibiotics such as vancomycin, colistin, nystatin, or trimethoprim to inhibit the growth of other bacteria and fungi that may contaminate the specimens.

One of the major applications of Mueller Hinton Agar (MHA) is for antibiotic susceptibility testing using the Kirby-Bauer disc diffusion method. This method is a simple and inexpensive way to determine how sensitive a bacterium is to different antibiotics. The Kirby-Bauer method involves the following steps :

• A pure culture of the bacterium to be tested is grown overnight in a liquid medium.

• A sterile cotton swab is dipped into the bacterial culture and used to inoculate a plate of MHA by streaking it over the entire surface of the agar.

• Small discs of filter paper impregnated with known concentrations of different antibiotics are placed on the surface of the inoculated agar plate using sterile forceps.

• The plate is incubated at 35°C for 16 to 18 hours.

• The diameter of the zone of inhibition around each disc is measured and compared with a standard chart to determine whether the bacterium is resistant, intermediate, or sensitive to each antibiotic.

The zone of inhibition is the clear area around the disc where the bacterial growth is inhibited by the antibiotic. The size of the zone depends on several factors, such as the diffusion rate of the antibiotic, the concentration of the antibiotic, the growth rate of the bacterium, and the thickness and pH of the agar. Therefore, it is important to use standardized conditions and quality control strains when performing this test.

The Kirby-Bauer method can help clinicians choose appropriate antibiotics for treating bacterial infections by identifying which antibiotics are effective against a specific bacterium. However, this method has some limitations, such as:

• It cannot be used for anaerobic bacteria, slow-growing bacteria, or capnophilic bacteria that require special growth conditions.

• It cannot provide quantitative information on the minimum inhibitory concentration (MIC) or minimum bactericidal concentration (MBC) of an antibiotic, which are more precise measures of antibiotic potency.

• It may be affected by drug inactivation, drug interactions, or bacterial resistance mechanisms that are not detected by this method.

Therefore, the Kirby-Bauer method should be used in conjunction with other methods and clinical judgment to guide antibiotic therapy.

References:

The Clinical and Laboratory Standards Institute (CLSI) is an organization that develops standards and guidelines for clinical and laboratory testing. One of the main functions of CLSI is to provide recommendations for antimicrobial susceptibility testing using the Kirby-Bauer disc diffusion method. These recommendations include the selection of appropriate media, quality control procedures, interpretation criteria, and reporting formats.

One of the key components of the CLSI recommendations is the establishment of zone diameter breakpoints for different antimicrobial agents and bacterial species. Zone diameter breakpoints are the values that define whether a bacterial isolate is resistant, intermediate, or susceptible to a given antimicrobial agent based on the size of the zone of inhibition around the disc. The zone diameter breakpoints are determined by correlating them with the minimum inhibitory concentration (MIC) breakpoints, which are the lowest concentrations of antimicrobial agents that inhibit the growth of bacteria in broth or agar dilution methods.

The zone diameter breakpoints are listed in the CLSI document M100, which is updated annually to reflect the latest scientific evidence and clinical relevance. The zone diameter breakpoints vary depending on the antimicrobial agent, the bacterial species, and sometimes the infection site or patient population. For example, the zone diameter breakpoints for ceftriaxone against Enterobacteriaceae are ≥23 mm for susceptible, 20-22 mm for intermediate, and ≤19 mm for resistant, while the zone diameter breakpoints for ceftriaxone against Streptococcus pneumoniae are ≥28 mm for susceptible, 25-27 mm for intermediate, and ≤24 mm for resistant.

The zone diameter breakpoints are intended to be used as a guide for clinical decision-making and not as absolute values. They may not apply to all situations or scenarios, and they may be modified by local or regional epidemiology, resistance mechanisms, pharmacokinetics, pharmacodynamics, or clinical outcomes. Therefore, it is important to consult with an infectious disease specialist or a clinical microbiologist when interpreting and reporting antimicrobial susceptibility test results.

Mueller Hinton Agar with 5% Sheep Blood is a modified version of Mueller Hinton Agar that contains 5% defibrinated sheep blood. This medium is recommended by the Clinical and Laboratory Standards Institute (CLSI) for antimicrobial susceptibility testing of Streptococcus pneumoniae and Haemophilus influenza using the disc diffusion method. These organisms are fastidious and require blood supplementation for optimal growth and reliable results.

Mueller Hinton Agar with 5% Sheep Blood is used for testing the susceptibility of S. pneumoniae to selected agents, such as chloramphenicol, erythromycin, ofloxacin, tetracycline, and vancomycin, as well as for screening the resistance to penicillin using oxacillin discs. The medium is also used for testing the susceptibility of H. influenzae to ampicillin, amoxicillin-clavulanate, cefaclor, cefixime, cefuroxime, chloramphenicol, and trimethoprim-sulfamethoxazole.

The addition of sheep blood to Mueller Hinton Agar enhances the growth of S. pneumoniae and H. influenzae and provides a source of X and V factors for H. influenzae. The blood also facilitates the detection of beta-lactamase production by H. influenzae, which appears as a pink halo around the disc. The medium has low levels of thymidine and thymine, which are inhibitors of sulfonamides and trimethoprim, and controlled levels of calcium and magnesium ions, which affect the activity of some antibiotics.

Mueller Hinton Agar with 5% Sheep Blood should be prepared and used according to the CLSI guidelines and quality control procedures. The medium should be inoculated with a standardized suspension of the organism and incubated in a CO2-enriched atmosphere for 20-24 hours at 35°C. The zones of inhibition around the discs should be measured and interpreted according to the CLSI criteria.

The Clinical and Laboratory Standards Institute (CLSI) is an organization that develops standards and guidelines for clinical and laboratory testing. CLSI has recommended Mueller Hinton Agar (MHA) as the standard medium for antibiotic susceptibility testing using the disc diffusion method. MHA has been selected by the CLSI for several reasons:

• It demonstrates good batch-to-batch reproducibility for susceptibility testing. This means that different lots of MHA from the same or different manufacturers produce consistent results when tested with the same quality control strains and antimicrobial discs.

• It is low in sulfonamide, trimethoprim, and tetracycline inhibitors. These are substances that can interfere with the activity of some antibiotics by blocking their uptake or metabolism by bacteria. MHA has a low concentration of thymidine and thymine, which are inhibitors of sulfonamides and trimethoprim, and a low concentration of para-aminobenzoic acid (PABA), which is an inhibitor of sulfonamides.

• It supports the growth of most non-fastidious bacterial pathogens. Non-fastidious bacteria are those that do not require special nutrients or conditions to grow. MHA provides adequate amounts of nitrogen, carbon, amino acids, vitamins, and minerals for the growth of the most common bacteria that cause infections in humans.

• Much data and much experience regarding its performance have been recorded. MHA has been widely used and studied for antibiotic susceptibility testing since the 1960s. Many publications and reports have documented its reliability and validity for different types of bacteria and antibiotics.

These reasons make MHA a suitable and preferred medium for antibiotic susceptibility testing using the disc diffusion method. However, MHA may not be appropriate for some fastidious or anaerobic bacteria, which may require special media or supplements to grow. MHA may also show variation in the concentration of divalent cations, such as calcium and magnesium, which can affect the results of some antibiotics, especially aminoglycosides, tetracyclines, and colistin.

Mueller Hinton Agar (MHA) is a widely used medium for antibiotic susceptibility testing using the disk diffusion method, also known as the Kirby-Bauer method. This method involves placing small disks impregnated with different antibiotics on the surface of the agar and measuring the zone of inhibition around each disk to determine the susceptibility of the bacterial isolate to that antibiotic.

MHA has several properties that make it suitable for this purpose:

• It is a non-selective, non-differential medium. This means that almost all organisms plated on it will grow, and there are no indicators or additives that could interfere with the interpretation of the results.

• It contains starch. Starch is known to absorb toxins released from bacteria so that they cannot interfere with antibiotics. It also mediates the rate of diffusion of the antibiotics through the agar.

• It is a loose agar. This allows for better diffusion of the antibiotics than most other plates. A better diffusion leads to a truer zone of inhibition.

• It shows acceptable batch-to-batch reproducibility for susceptibility testing. This means that the results are consistent and reliable across different batches of the medium.

• It is low in sulfonamide, trimethoprim, and tetracycline inhibitors (i.e., the concentration of inhibitors thymidine and thymine is low in MHA). These inhibitors can reduce the activity of some antibiotics by competing with them for binding sites on bacterial enzymes or DNA.

• It has a controlled pH and cation concentration. The pH of MHA is adjusted to neutral at 25°C, and the levels of calcium and magnesium ions are kept low. These factors can affect the activity of certain antibiotics, especially aminoglycosides, quinolones, macrolides, tetracyclines, and colistin.

These properties make MHA an ideal medium for antibiotic susceptibility testing, as it provides a reliable and consistent substrate for bacterial growth and antibiotic diffusion. However, MHA may not be suitable for some fastidious or anaerobic organisms, which may require supplementation of blood or other nutrients. MHA may also need to be modified for some specific tests, such as adding sheep blood for testing Streptococcus pneumoniae and Haemophilus influenza.

Mueller Hinton Agar media contains Beef Extract, Acid Hydrolysate of Casein, Starch, and Agar. Beef Extract and Acid Hydrolysate of Casein provide nitrogen, vitamins, carbon, amino acids, sulfur, and other essential nutrients for bacterial growth. Starch acts as a colloid and is added to absorb any toxic metabolites produced by bacteria. Starch also mediates the rate of diffusion of the antibiotics through the agar. Agar is the solidifying agent.

The principle of Mueller Hinton Agar is based on the fact that it is a non-selective, non-differential medium that supports the growth of most non-fastidious bacterial pathogens. It is also low in sulfonamide, trimethoprim, and tetracycline inhibitors, such as para-aminobenzoic acid (PABA), thymidine, and thymine, which can interfere with the activity of these antibiotics. The levels of calcium and magnesium ions are also controlled to avoid interference with the activity of certain antibiotics, such as aminoglycosides, tetracyclines, and colistin.

Mueller Hinton Agar is suitable for testing the susceptibility of microorganisms to sulfonamides and trimethoprim by the disk diffusion method, as these antibiotics are inactivated by PABA and thymidine, respectively. The disk diffusion method involves placing small disks impregnated with different antibiotics on the surface of the agar and measuring the zone of inhibition around each disk to determine the susceptibility of the bacterial isolate to that antibiotic. The zone of inhibition depends on the diffusion rate of the antibiotic, the degree of susceptibility of the organism, and the growth rate of the organism. Mueller Hinton Agar allows for better diffusion of the antibiotics than most other media, resulting in a truer zone of inhibition.

Mueller Hinton Agar has been selected by the Clinical and Laboratory Standards Institute (CLSI) for several reasons: it demonstrates good batch-to-batch reproducibility for susceptibility testing; it has a low content of inhibitors that could affect the results; and it has many data and much experience regarding its performance recorded.

Mueller Hinton Agar (MHA) is a microbiological growth medium that consists of the following ingredients per liter of distilled water :

• Beef extract: 2.0 g

• Acid hydrolysate of casein: 17.5 g

• Starch: 1.5 g

• Agar: 17.0 g

The final pH of the medium is adjusted to 7.3 ± 0.1 at 25°C.

Beef extract and acid hydrolysate of casein provides nitrogen, vitamins, carbon, amino acids, sulfur, and other essential nutrients for bacterial growth. Starch acts as a colloid and absorbs toxic metabolites that may interfere with antibiotic activity. Starch also hydrolyzes to dextrose, which serves as an energy source. Agar is the solidifying agent that gives the medium a firm consistency.

Some modifications of MHA may include additional ingredients, such as sheep blood (5%) and nicotinamide adenine dinucleotide (NAD), for susceptibility testing of certain bacteria, such as Streptococcus and Campylobacter species. These supplements enhance the growth and hemolysis of these bacteria and facilitate the interpretation of the results.

The composition of MHA is carefully controlled to ensure optimal performance for antibiotic susceptibility testing. The levels of calcium and magnesium ions, which can affect the activity of some antibiotics, are kept low. The levels of thymidine and thymine, which can inhibit the action of sulfonamides and trimethoprim, are also minimized. The medium is free of inhibitors that could interfere with bacterial growth or antibiotic diffusion.

MHA is a nutrient-rich and non-selective medium that supports the growth of most non-fastidious bacterial pathogens. It is also a non-differential medium that does not distinguish between different types of bacteria based on their appearance or biochemical reactions. Therefore, MHA is suitable for testing a wide range of antibiotics against various bacterial isolates using the disk diffusion method[^1 ^][ ^2 ^][ ^4 ^].

To prepare Mueller Hinton Agar (MHA), you will need the following ingredients :

• Beef extract: 2.0 g

• Acid hydrolysate of casein: 17.5 g

• Starch: 1.5 g

• Agar: 17.0 g

• Distilled water: 1 liter

The steps for preparing MHA are as follows :

1. Suspend 38 g of the medium in 1 liter of distilled water and mix thoroughly.

1. Heat with frequent agitation and boil for 1 minute to completely dissolve the medium.

1. Autoclave at 121°C for 15 minutes.

1. Cool to room temperature.

1. Pour cooled MHA into sterile Petri dishes on a level, horizontal surface to give uniform depth. For disk diffusion tests, pour accurately measured volumes of molten agar into plates:

• 60 to 70 ml/150-mm plate

• 25 to 30 ml/100-mm plate

1. Eliminate bubbles on the molten agar surface by quickly (and carefully) passing the flame from a Bunsen burner over the agar.

1. Allow plates to solidify at room temperature with plate lids slightly ajar.

1. Check prepared MHA to ensure the final pH is 7.3 ±1 at 25°C. Note: If the pH is <7.2, certain drugs will appear to lose potency (aminoglycosides, quinolones, macrolides), while other agents may appear to have excessive activity (tetracycline). If the pH is >7.4, the opposite results may occur.

1. Store prepared plates at 2 to 8°C in tightly sealed packages.

Note: The correct depth of MHA for disk diffusion testing is critical. Failure to dispense accurate volumes may result in agar that is too thin (often yielding false-susceptible results) or too thick (often yielding false-resistant results).

The results on Mueller Hinton Agar (MHA) depend on the type and concentration of the antibiotic used, the bacterial species and strain tested, and the size and density of the inoculum. The results are usually interpreted by measuring the diameter of the zone of inhibition around each antibiotic disk and comparing it with the standard values provided by the Clinical and Laboratory Standards Institute (CLSI) or other authoritative sources.

The zone of inhibition is the clear area around the disk where the bacterial growth is inhibited by the antibiotic. The size of the zone reflects the diffusion rate of the antibiotic through the agar, the degree of susceptibility of the bacteria to the antibiotic, and the growth rate of the bacteria. The zone size may also be affected by factors such as agar thickness, pH, temperature, incubation time, and the presence of inhibitors or enhancers.

The zone diameters are measured using a ruler or a caliper and recorded in millimeters. The measurements should be taken from the bottom of the plate and include the diameter of the disk (6 mm). The measurements should be rounded off to the nearest whole millimeter.

The zone diameters are then compared with the interpretive criteria provided by CLSI or other sources for each bacterial species and antibiotic combination. The criteria are based on extensive studies and clinical data that correlate the zone diameters with the minimum inhibitory concentrations (MICs) and clinical outcomes. The criteria classify the results into three categories: susceptible (S), intermediate (I), or resistant (R).

• Susceptible (S) means that the isolate is likely to be inhibited by the usually achievable concentrations of the antibiotic when the dosage is appropriate for the site of infection.

• Intermediate (I) means that the isolate may be susceptible if a higher dosage or higher concentration at the site of infection can be achieved or if there is no alternative drug available. It also indicates a buffer zone to prevent small errors in testing from causing major discrepancies in interpretation.

• Resistant (R) means that the isolate is not likely to be inhibited by the usually achievable concentrations of the antibiotic, and other therapy should be selected.

The interpretation of the results should also take into account other factors such as the patient`s condition, site of infection, pharmacokinetics and pharmacodynamics of the antibiotic, potential adverse effects, drug interactions, cost, and availability.

The results of Mueller Hinton Agar (MHA) can provide valuable information for guiding empirical therapy, monitoring resistance trends, detecting emerging resistance mechanisms, and evaluating new antibiotics. However, they have some limitations and should not be used alone for definitive diagnosis or treatment decisions. Some limitations are:

• Not all bacterial species or antibiotics can be tested by this method. Some fastidious or anaerobic bacteria may require special media or conditions for growth. Some antibiotics may have poor diffusion or stability in agar or may be affected by inhibitors or enhancers in the medium.

• The results may not reflect the actual situation in vivo, where factors such as host immunity, biofilm formation, tissue penetration, drug metabolism, and drug distribution may influence the efficacy of antibiotics.

• The results may vary depending on the quality control and standardization of the testing procedure. Factors such as inoculum preparation, disk potency, agar composition and thickness, incubation time and temperature, and measurement technique may affect the accuracy and reproducibility of results.

• The results may not account for some resistance mechanisms that are not detected by the disk diffusion method. For example, some bacteria may produce enzymes that inactivate antibiotics (e.g., beta-lactamases) or may have efflux pumps that expel antibiotics from cells. These mechanisms may require special tests or molecular methods to confirm.

Therefore, it is important to follow the guidelines and recommendations for performing and interpreting disk diffusion tests on Mueller Hinton Agar (MHA) and to consult with experts or reference laboratories when in doubt. Disk diffusion tests on MHA are useful screening tools for antimicrobial susceptibility testing, but they should be supplemented by other methods, such as MIC determination or molecular typing, when necessary.

Mueller Hinton Agar (MHA) has several uses in microbiology, mainly related to the isolation and susceptibility testing of bacterial pathogens. Some of the common uses of MHA are:

• Antibiotic susceptibility testing using the Kirby-Bauer disc diffusion method: This is the most widely used application of MHA, as it allows for a simple and reliable assessment of the sensitivity or resistance of bacterial isolates to various antibiotics. MHA is suitable for testing a broad range of antibiotics, as it has low levels of calcium and magnesium ions, which can interfere with some antibiotics, and it is free of inhibitors that can affect bacterial growth. MHA also contains starch, which absorbs toxins released by bacteria and mediates the diffusion rate of antibiotics through the agar. The zone of inhibition around each antibiotic disc indicates the susceptibility of the bacterial isolate to that antibiotic.

• Isolation and maintenance of Neisseria and Moraxella species: MHA can also be used to culture these fastidious gram-negative bacteria, which are often associated with respiratory infections. MHA provides adequate nutrients and pH for their growth.

• Food testing for aerobic and facultative anaerobic bacteria: MHA can be used to detect and enumerate bacteria that are commonly found in food products, such as Escherichia coli, Salmonella, Staphylococcus aureus, and Listeria monocytogenes. MHA is a nutrient-rich and non-selective medium that supports the growth of most non-fastidious bacteria.

• Rapid presumptive identification of Candida albicans: MHA can be used as an alternative method for the germ tube test, which is a common technique for identifying C. albicans, a yeast that can cause oral thrush and vaginal infections. MHA can induce the formation of germ tubes by C. albicans within 2 hours, while other media may take longer or fail to do so.

Mueller Hinton Agar is a widely used medium for antimicrobial susceptibility testing, but it has some limitations that should be considered:

• It is recommended for susceptibility testing of pure cultures only. Mixed cultures may interfere with the interpretation of the results.

• Inoculum density may affect the size of the microbial growth zone of inhibition. Heavy inoculum may result in smaller zones, or too less inoculum may result in larger zones.

• Fastidious organisms may not grow on this medium and may require supplementation of blood or other nutrients. However, some supplements may have an inactivating effect on the antimicrobial agents tested.

• Obligate anaerobes, slow-growing organisms, and capnophiles may not be suitable for the disk diffusion method using MHA, as they require special conditions for growth, and drug inactivation may occur due to prolonged incubation times.

• Variation in the concentration of divalent cations, primarily calcium, and magnesium, affects the result of aminoglycoside, tetracycline, and colistin test with Pseudomonas aeruginosa isolates.

• Some organisms may produce pigments or hemolysis that may obscure the zone edges and make them difficult to read.

Therefore, it is important to follow the standard protocols and quality control procedures when using MHA for antimicrobial susceptibility testing and to consult the CLSI guidelines for specific recommendations and interpretations.

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