Mueller Hinton Agar (MHA)

Mueller Hinton Media contains Beef Extract, Acid Hydrolysate of Casein, Starch and Agar. Beef Extract and Acid Hydrolysate of Casein provide nitrogen, vitamins, carbon, amino acids, sulphur and other essential nutrients. Starch is added to absorb any toxic metabolites produced. Starch hydrolysis yields dextrose, which serves as a source of energy. Agar is the solidifying agent. Final pH 7.3 ± 0.1 at 25ºC

Composition of Mueller Hinton Agar (MHA)

Ingredients	Gms/Litre
Beef extract	2.0
Acid hydrolysate of casein	17.5
Starch	1.5
Agar	17.0

 Uses of MHA

1. The major use of Mueller Hinton Agar is for antimicrobial susceptibility testing. It has become the standard medium for the  Kirby-Bauer method

2. It can be used to cultivate Neisseria

3. It is specified in FDA Bacteriological Analytical Manual for food testing, and procedures commonly performed on aerobic and facultative anaerobic bacteria.

Why MHA is used for antibiotic susceptibility testing?

1. It is a non-selective, non-differential medium. This means that almost all organisms plated on here will grow.

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

3. 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.

4. MHA shows acceptable batch-to-batch reproducibility for susceptibility testing.

5. MHA is low in sulfonamide, trimethoprim, and tetracycline inhibitors (i.e. concentration of inhibitors thymidine and thymine is low in MHA).

6. It supports the growth of most non-fastidious bacterial pathogens.

Limitations of MHA

1. Numerous factors can affect results: inoculum size, rate of growth, medium formulation and pH. Strict adherence to protocol is required to ensure reliable results.

2. Drug inactivation may result from the prolonged incubation times required by slow growers.

3. This medium is recommended for susceptibility testing of pure cultures only. Inoculum density may affect the zone size.

4. Heavy inoculum may result in smaller zones or too less inoculum may result in bigger zones. 

5. Fastidious organisms may not grow on this medium and may require supplementation of blood. 

6. As antimicrobial susceptibility is carried with antibiotic disc, proper storage of the disc is desired which may affect the potency of the disc. 

Bombay blood group

 The rare, Bombay blood group was first discovered in Mumbai ( Bombay) in 1952 by Dr Y M Bhende. Each red blood cell has antigen over its surface, which helps determine which group it belongs to. In addition to the common A, B, D antigens in the positive blood group people, H antigen is also seen in all blood groups. Thus, in the AB positive blood group,  antigens A, B, D and H are found. A positive will have A, D and H antigens; B positive will have B, D and H antigens. O positive lack A, B but have D antigen. In the Bombay blood group, also called hh, is deficient in expressing antigen H, meaning the RBC has no antigen H. Thus, in hh, there are no A or B antigens, so they are similar to O group. But they alos lack H antigen, hence called Bombay blood group. Could be D positive or negative.

Blood type A is the most ancient, and it existed before the human species evolved from its hominid ancestors

Rarest blood type?

• AB-negative (. 6 percent)
• B-negative (1.5 percent)
• AB-positive (3.4 percent)

O+ is the most frequently occurring blood type and is found in 37 percent of the population. O- is found in six percent of the population

O positive donors who are CMV negative are known as Heroes for Babies at the Red Cross because it is the safest blood for transfusions for immune deficient newborns

Of the eight main blood types, people with type O have the lowest risk for heart disease. People with types AB and B are at the greatest risk

• People with A and AB blood have the highest rates of stomach cancer.

Type O individuals may be more likely to develop peptic ulcers caused by Helicobacter pylori bacterium than other blood types, and that type O women may have more risk of fertility problems

The golden blood type or Rh null blood group contains no Rh antigens (proteins) on the red blood cell (RBC). This is the rarest blood group in the world, with less than 50 individuals having this blood group

Rh incompatibility - If a woman who is Rh negative and a man who is Rh positive have a baby, the fetus may have Rh-positive blood, inherited from the father. Rh incompatibility usually isn't a problem if it's the mother's first pregnancy because the baby's blood does not normally enter the mother's circulatory system. During the birth, the mother's and baby's blood can mix. The mother's body recognizes the Rh protein as a foreign substance and begin making antibodies  against the Rh protein.Rh antibodies are harmless until the mother's second or later pregnancies. If she is next carrying another Rh-positive child, her Rh antibodies will recognize the Rh proteins on the surface of the baby's blood cells as foreign. Her antibodies will pass into the baby's bloodstream and attack those cells. This can make the baby's red blood cells swell and rupture. This is known as hemolytic or Rh disease of the newborn. 

Petroleum degradation- Alicyclic Hydrocarbons

 Alicyclic Hydrocarbons

Alicyclic Hydrocarbons with no terminal methyl groups. Eg., Cyclohexane 

Cyclohexane is acted upon by a monooxygenase forming an alicyclic alcohol, cyclohexanol, which is dehydrogenated to form cyclohexanone (ketone). Cyclohexanone is further oxidised to form a lactone. Lactone is oxidised to an alcohol, aldehyde and then a dicarboxylic acid. Further metabolism is by β oxidation.

Microorganisms are found to perform these reactions and grow on cyclohexane. More frequently however, microorganisms are unable to lactonize and open the ring. Commensalism and cometabolism thus play an important role in the biodegradation of Alicyclic Hydrocarbons

Alkyl benzyl sulphonates/ Alkyl benzene sulphonates(ABS)

 Alkyl benzyl sulphonates/ Alkyl benzene sulphonates (ABS)

•  Main components of anionic detergents-surface active with polar (water soluble) sulphate and nonpolar alkyl end 

• Aid in emulsification of fatty substances and cleaning occurs while these molecules make a monolayer around lipophilic droplets or particles.

• Their molecules orient with their nonpolar end towards the lipophilic substance and the sulphonate end towards water.

• Nonlinear alkylbenzene sulphonates or Branched alkylbenzene sulfonates (BAS) are recalcitrants and resistant to biodegradation and cause foaming problems in sewage treatment and  in the rivers.

• ABS are easier to manufacture and bear superior detergent properties but the methyl branching of alkyl chain in them interferes with biodegradation of ABS,  because tertiary carbon atom blocks the normal β-oxidation.

• Nonlinear alkylbenzyl sulphonates or Branched alkylbenzene sulfonates (BAS) were replaced with linear alkylbenzene sulfonates (LAS) during the 1960s due to environmental concerns

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LAS (linear alkylbenzyl sulphonates) are biodegradable

• It is biodegraded rapidly under aerobic conditions within weeks- oxidative degradation initiates at the alkyl chain.

• Under anaerobic conditions it degrades very slowly or not at all, accumulates in sewage sludge, but once oxygenated, it will rapidly degrade

• Thus, BAS to LAS conversion is a story where a synthetic molecule was redesigned to remove obstacles to biodegradation, while preserving the other useful characteristics of the compound.

Gram-negative cell walls

•        Much more complex than gram-positive walls

•        The thin peptidoglycan layer - not more than 5 to 10% of the wall weight

•        (E. coli - 2 nm thick; only one or two layers or sheets of peptidoglycan)

•        The outer membrane lies outside the thin peptidoglycan layer

•         The most abundant membrane protein is Braun’s lipoprotein, a small lipoprotein joined to the peptidoglycan and extends to the outer membrane by its hydrophobic end 

                        

•        Outer membrane contains large, complex lipopolysaccharides (LPSs) and consist of three parts: (1) lipid A, (2) the core polysaccharide, and (3) the O side chain.

•        The lipid A region buried in the outer membrane and part of the LPS molecule projects from the surface

•        The core polysaccharide is joined to lipid A; In Salmonella, it is constructed of 10 sugars, many of them unusual in structure.

•        The O side chain or O antigen is a polysaccharide chain extending outward from the core; varies in composition between bacterial strains

•        O side chains readily recognized by host antibodies- gram- negative bacteria overcome host defenses by rapidly changing the nature of their O side chains to avoid detection.

•        Antibody interaction with the LPS before reaching the outer membrane protect the cell wall from direct attack

•        LPS contributes to the negative charge on the bacterial surface (core polysaccharide usually contains charged sugars and phosphate )

•        stabilizes membrane structure

•        Lipid A is toxic; LPS can act as an endotoxin; gram-negative bacterial infections

•        Protective barrier- prevents or slows the entry of bile salts, antibiotics, and other toxic substances

•        Outer membrane more permeable than the plasma membrane; permits the passage of small molecules like glucose and other monosaccharides

•        Presence of special porin proteins (Larger molecules such as vitamin B12 must be transported across the outer membrane by specific carriers )

•        The outer membrane also prevents the loss of constituents like periplasmic enzymes

•        A space is seen between the plasma membrane and the outer membrane in gram- negative bacteria, and between the plasma membrane and cell wall in gram- positive bacteria-periplasmic space containing periplasm

•        The periplasmic space contains many proteins, for example, hydrolytic enzymes, transport proteins etc

•         The periplasmic space also contains enzymes involved in peptidoglycan synthesis and the modification of toxic compounds that could harm the cell.

Recalcitrant halocarbons

 

•   Common xenobiotic pollutants
•      These compounds contain different numbers of halogen (e.g., CI, Br, F (fluorine), I) atoms in the place of H atoms.
•     C-Halogen bond highly stable-cleavage requires considerable energy.
• Halocarbons are chemically and biologically very stable- persistent compounds, cause environmental pollution
• Two classes-

• Recalcitrant Halocarbons

  • Haloalkyls- Haloalkyl propellants & solvents
  • Haloaromatics- Halobenzenes, halophenols & halobenzoates

 

Haloalkyl propellants & solvents

• ·         C₁-C₂ alkanes in which all or almost all H atoms are replaced by halogens like chlorine, fluorine etc
• ·         They are used as solvents (chloroform, CHCI₃), as propellants in spray cans of cosmetics, paints etc., in condenser units of cooling systems (Freons, CCI₃F, CCl₂F₂, CClF₃, CF₄),
• ·         Chloroform, CCl₄, dichloromethane, dichloro, trichloro, tetra chloro ethenes are industrial and cleaning solvents-  Spilled halocarbon propellants and solvents contaminate ground water
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• ·         C₁-C₂ halo alkanes like chloroform, freons etc. are volatile and escape into the stratosphere where they destroy the protective ozone (O₃) layer leading to increased UV radiation-increased incidence of skin cancer and mutagenesis
• ·         Phasing out of halocarbons as aerosol propellants now in practice-research to find out suitable alternatives as refrigerants-Freons gradually being replaced by C₂ based halocarbons containing Cl₂ in place of Fluorine- photo chemically less destructive to the ozone layer, though biodegradability continues to be problem
• ·    Dehalogenation of organic compounds thermodynamically favored under anaerobic conditions by dehalogenases (reductive dehalogenation)
• ·       Tetrachloro/perchloro ethene undergoes stepwise dechlorination and is finally converted to vinyl chloride (toxic) and finally, ethane by methanogenic bacterial consortia
• ·    Aerobically, dichloro methane (DCM) can be utilised as carbon source by Pseudomonas. Dichloro ethene is acted upon by microbial consortium. Tricholoro ethene (TCE) is acted upon by methane monooxygenase to form TCE epoxide which further is converted to formate + CO, glyoxylate and dichloro acetate. TCE can also be converted to unstable formic acid and glyoxylic acid by toluene dioxygenase of Pseudomonas
• ·   Chloro and bromomethane are acted upon by Methylococcus capsulatus to yield formaldehyde, dichloro methane, trichloromethane

(contd..)

Gram-Positive Cell Wall

•        Outer boundary of the cell; one of the most important parts of a Procaryotic cell

•         Most bacteria have strong walls that give them shape and protect them from osmotic lysis (Exception- mycoplasmas and some Archaea) 

•        Wall shape and strength is primarily due to peptidoglycan

•        The cell walls of many pathogens contribute to their pathogenicity

•         The wall can protect a cell from toxic substances and is the site of action of several antibiotics

Peptidoglycan or Murein

•        polymer composed of identical subunits

•        alternating sugar derivatives-

•         N-acetylglucosamine (NAG) & N-acetylmuramic acid (NAM)

•        Connected to the carboxyl group of N-acetylmuramic acid is a peptide chain of 4 alternating D- and L-amino acids (D-glutamic acid, L/D-alanine, diaminopimelic acid (not found in proteins) etc. (L-lysine, in some)

•        The presence of D-amino acids protects against attack by most peptidases

•        Chains of linked peptidoglycan subunits are joined by cross- links between the peptides

•        Cross-linking results in a dense, interconnected network of peptidoglycan

•        strong enough - retain the shape and integrity

•        elastic and stretchable

•        porous, for molecules to penetrate

Gram-Positive Cell Walls

•        Thick, homogeneous cell wall of gram-positive bacteria composed primarily of peptidoglycan

•        Contain large amounts of teichoic acids (polymers of glycerol or ribitol joined by phosphate groups)

                                                Gram-Positive Cell Wall

•        Teichoic acids - connected by a covalent bond to N-acetylmuramic acid of the peptidoglycan or to plasma membrane lipids (lipoteichoic acids)

•   Teichoic acids extend to the surface of the peptidoglycan-negatively charged- give the gram-positive cell wall its negative charge.

•    Important in maintaining the structure of the wall.

•        Teichoic acids - not present in gram-negative bacteria.