FAQ in bacterial staining techniques

How are staining techniques classified?

• Simple stain: where only one stain is used and all bacteria are stained similarly. Eg: Methylene blue, dilute carbol fuchsin
• Differential staining: where different bacteria stain differently to a common staining technique depending on their physiological properties. Eg: Gram’s stain and Acid fast staining
• Special stain: where structures of bacteria like spores, granules, capsule etc are demonstrated. Eg: silver impregnation technique for demonstration of spirochetes, Feulgen stain for demonstration of nucleus, Sudan black stain for demonstration of lipid vacuoles, Ryu’s stain for demonstration of flagella, Albert’s stain for demonstration of metachromatic granules.
• Negative staining: where the background is stained with an acidic dye such as India ink or Nigrosin. Used for demonstration of capsules.

How are stains classified?

Stains are classified based on the pH of their chromophore (color bearing ion) into acidic, basic and neutral. Acidic dyes have anionic chromophore eg., sodium+ eosinate-. Basic dyes have cationic chromophore eg., methylene blue+ chloride-. Acidic dyes combine more strongly with cytoplasmic components of bacteria, especially the nucleus that is basic in nature. Neutral dyes have both acidic and basic component that nullify each other. They are Romanowsky’s stain and are used in staining parasitic forms. Stains can be either natural (eg: carmine and hematoxylin) or coal-tar derivatives /aniline stains (eg: methylene blue, crystal violet). Supravital (cells removed from the body) and intravital (cells still a part of the body).

What is polychrome methylene blue?

Loeffler’s methylene blue solution treated with Potassium hydroxide turns into Polychrome methylene blue after prolonged storage with shaking. Used in McFadyean’s reaction for Bacillus anthracis in blood films and demonstration of metachromatic granules of Corynebacterium diphtheriae.

Who invented Gram stain?

Hans Christian Gram invented this stain in 1884. The original formulation was Aniline Gentian violet, Lugol’s iodine, absolute alcohol and Bismark brown.

Which are the theories of Gram staining?

• Cell wall theory: Cell wall of Gram positive bacteria are 40 times thicker than those of Gram negative cells, hence they are thought to help retain the dye-iodine complex.
• Lipid Content Theory: Cell envelope of Gram negative bacteria contains an additional membrane (outer membrane), hence containing more lipids than Gram positive bacteria. Acetone or alcohol dissolves the lipid thus forming large pores in Gram negative bacteria through which the dye-iodine complex leaks out. Alcohol/acetone dehydrates Gram positive bacteria shrinking the cell wall and the closing the pores.
• Magnesium Ribonucleate Theory: A compound of magnesium ribonucleate and basic protein concentrated at the cell membrane helps Gram positive bacteria retain the primary dye. Gram negative bacteria do not possess this substance.
• Cytoplasmic pH Theory: The cytoplasm of Gram positive bacteria are said to be more acidic (2) than those of Gram negative ones (3). Hence the dye is said to bind with more affinity to Gram positive cells.

Which part of the bacteria actually gets stained?

It is the cytoplasm (especially the nucleic acid) that gets stained and not the cell wall. Presence of an intact cell wall is important for retaining Gram positivity. Cell wall deficient forms such as Mycoplasma and L forms are Gram negative.

Which are the bacteria or bacterial component that can’t be stained by Gram stain?

• Extremely slender bacteria such as Treponema
• Cells containing waxy substances impermeable to stain such as Mycobacteria
• Minute intracellular bacteria such as Chlamydia and Rickettsia
• Cell organelles such as capsule, spore, flagella etc

Which are the alternatives used in Gram stain?

• Primary stain: Crystal violet, Methyl violet and Gentian violet
• Mordant: Gram’s iodine, rarely Lugol’s iodine
• Decolorizer: Alcohol, acetone, acteone-alcohol mixture (1:1)
• Counterstain: Dilute carbol fuchsin, safranin, neutral red, (Sandiford stain for Gonococci)

Which are the positive and negative controls for Gram stain?

• Postive control: Staphylococci
• Negative control: E.coli, pus cells

What are the conditions when Gram positive bacteria can appear Gram negative?

• When over-decolourized by either prolonged exposure to decolourizer or using acetone alone.
• When cell wall gets damaged by exposure to lysozyme or cell wall acting antibiotics such as Penicillin.
• Old cultures, where cell wall is weakened or action of autolytic enzymes
• Those bacteria that are phagocytosed, where cell wall is acted upon by lysosomal contents

Which is the more important step in Gram stain?

Decolourization is the most important step as this step differentiates between Gram positive and Gram negative bacteria. Over-decolourization can result in Gram positive bacteria appearing Gram negative and under-decolourization can result in Gram negative bacteria appearing Gram positive.

What are the applications of Gram staining?

• Rapid presumptive diagnosis of diseases such as bacterial meningitis
• Selection of empirical antibiotics based on Gram stain finding
• Selection of suitable culture media based on Gram stain finding
• Screening of quality of clinical specimens, such as sputum that should contain many pus cells and few epithelial cells
• Counting of bacteria
• Appreciation of morphology and types of bacteria in a clinical specimen

Name a fungus that is Gram positive?

Candida sps

What are the various modifications of Gram stain?

• Kopeloff and Beerman’s (Primary stain: Methyl violet, decolourizer: acetone or alcohol-acetone mixture 1:1)
• Jensen’s (Primary stain: Methyl violet, decolourizer: absolute alcohol, counterstain: Neutral red)
• Preston and Morrell’s (Primary stain: crystal violet, decolourizer: iodine-acetone)
• Weigert’s (Primary stain: Carbol gentian violet, decolourizer: Aniline-xylol). This is used to stain tissue sections.

What is acid fast staining?

Certain bacteria or their structures have the ability to retain the primary dye (strong carbol fuchsin) and resist decolourization by weak mineral acids such as H2SO4, HCl. Such bacteria or their structure are termed acid fast and this property is termed acid fastness. There are two types of acid fast staining, the hot method and the cold method. The hot method (Ziehl-Neelsen) involves heating the slide while the cold methods such as Kinyoun’s and Gabbett’s do not involve heating the slide.

Who introduced Acid fast staining?

Ehrlich in 1882 discovered acid fastness. The original method involved staining with aniline-gentian violet and decolourization with strong nitric acid. It was later improved by Ziehl and Neelsen.

Why are Mycobacteria acid fast?

The cell walls of Mycobacteria are made up of waxy substance, Mycolic acid that is relatively impermeable to ordinary staining techniques. But, by application of heat and a mordant (phenol), the cell can be stained. The purpose of heating is to soften the waxy material of the cell wall and allow the stain to enter the cell. Basic fuchsin is more soluble in phenol and phenol is a better solvent for lipids and waxes.

What are the components of Ziehl-Neelsen stain?

• Primary stain: Strong Carbol Fuchsin (contain Basic fuchsin and Phenol)
• Decolourizer: 20% sulphuric acid
• Counterstain: Loeffler’s Methylene blue or 1% Malachite green, Picric acid for color-blind workers

What is acid-alcohol decolourizer?

3% HCl in 95% alcohol (methylated spirit). This is useful in differentiating saprophytic Mycobacteria from pathogenic Mycobacteria. Pathogenic Mycobacteria are both acid and alcohol fast but saprophytic Mycobacteria are only acid-fast. Saprophytic Mycobacteria can get declourized by alcohol. 95% alcohol can be used as a secondary decolorizer after decolourizing with acid. Especially used in staining smears prepared from urine that may contain Mycobacterium smegmatis.

Which are the various dilutions of sulfuric acid used?

• Mycobacterium leprae - 5% H2SO4
• Oocysts of Cryptosporidium, Isospora - 1% H2SO4
• Tissue sections containing Actinomyctes, Nocardia - 1% H2SO4
• Cultures of Nocardia - 0.5% H2SO4
• Bacterial spores - 0.25-0.5% H2SO4

What are cold methods of acid fast staining?

The two methods namely Kinyoun’s and Gabbett’s don’t involve heating of slides, hence called cold methods. Heating is substituted by increased concentration of phenol and prolonging the duration of staining. Kinyoun's method is favoured for detection of Cryptosporidium oocysts in fecal samples. Gabbett’s method has decolourizer and counterstain in one solution.

Why should the slide be flooded with strong carbol fuchsin?

For uniform distribution of heat, or else the slide may break.

What are the precautions to be taken while preparing or observing smears for AFB?

• A new slide must be used for every specimen, because scratch marks may give false positive.
• A uniform smear from thick portion of the sputum must be made.
• Staining jars should not be used to staining smear as there is risk to cross contamination.
• Fresh blotting paper must be used for each smear for drying the slide to prevent transfer from one slide to another.

How to interpret the smear?

At least 100 oil immersion fields must be viewed before declaring the smear as negative. The sensitivity of smear is low because it requires the presence of 104 bacilli/ml to be smear positive. If the number of bacilli is less than this, the chances of detecting them are less. In such a case, the sample should be subjected to concentration techniques such as Petroff’s method. If the smear is positive for AFB, it should be counted/graded. Failure to detect any AFB does not rule tuberculosis. Grading of smears has prognostic value.

How is the smear graded?

Smears are graded depending on the number of bacilli seen.

• 3-9 bacilli/entire smear: +
• ≥10 bacilli/entire smear: ++
• ≥10 bacilli/in most oil immersion fields: +++

What other methods are available for staining Mycobacteria?

Sputum smears for Mycobacteria can be stained by fluorescent dyes such as Auramine and Rhodamine as they have affinity for mycolic acid in their cell walls. The fluorescent microscopy is useful in screening large number of specimens. Large area of smear can be quickly observed that too under high power dry objective.

What is beaded appearance of Mycobacteria?

Beaded appearance is used to describe the appearance of Mycobacteria when the cell doesn’t stain uniformly, showing stained and unstained regions. These forms are common in Mycobacterium tuberculosis while Mycobacterium bovis stains uniformly. Most saprophytic Mycobacteria stain uniformly.

What are metachromatic granules?

Metachromatic granules are polymetaphosphate reserves produced by Corynebacterium diphtheriae in nutritious medium. These granules are also known as Babes Ernst granules, Volutin granules, Polar bodies etc. They are called metachromatic granules because of they exhibit metachromasia, a property where the granules appear in a colour different from that of the dye used. When stained with polychrome methylene blue, they appear purple. They are produced in abundance in serum containing medium such as Loeffler’s serum slope.

Which are the ways to demonstrate these granules?

Albert’s stain, Neisser’s stain, Ponder’s stain and Pugh’s stain. They can be demonstrated as refractile bodies in wet mount or slightly more gram positive structures in Gram stain.

Why are the bacilli arranged at angles to each other?

The bacilli are arranged at angles to each other resembling English letter V or L or Chinese letter (cuneiform) pattern because the daughter cells don’t separate completely after cell division (binary fission).

What do Albert A(1) and B(2) solution contain?

Solution A(1) contains Toluidine blue, Malachite green, Glacial acetic acid and Alcohol while solution B(2) contains iodine and potassium iodide in distilled water.

FAQ in bacterial culture media

Name some simple media.

Peptone water, Nutrient broth and agar.

Name some enriched media

Blood agar, Chocolate agar, Dorset egg medium and Loeffler’s serum medium.

How are media made selective?

• Antibiotic - Thayer - Martin agar for Neisseria gonorrhoeae, CIN agar for Yersinia enterocolitica
• Dye - Brilliant green - in LJ media for Mycobacterium tuberculosis, Crystal violet blood agar for Group A Streptococcus,
• c) pH - TCBS and alkaline peptone water (pH >8) for Vibrio cholerae.
• d) Salt -Mannitol salt agar, with 7.5% NaCl for Staphylococcus aureus.
• e) Chemicals - Bile salts in Mac Conkey’s agar, Potassium tellurite in Hoyle’s medium and Mc Leod’s medium for Corynebacterium diphtheriae, Sodium azide agar for Enterococcus sp., Bismuth sulphite in Wilson and Blair agar for Salmonella typhi.
• f) Disinfectant - i) Cetrimide agar for Pseudomonas aeruginosa

Name some transport media.

Amies medium, Pikes medium, Stuart’s medium, Buffered glycerol saline and VR medium.

Name some enrichment broth.

Selenite F broth, Tetrathionate broth and Alkaline peptone water.

How are the media sterilized?

Most media are sterilized by autoclaving.

Which media are not autoclavable?

• Blood, serum, antibiotic and sugar containing media.
• Highly selective media such as Wilson & Blair’s, TCBS etc.

How are media solidified?

By adding 2 - 3% agar.

What is the percentage of blood used in Blood agar?

5% defibrinated sheep blood.

What is the melting and solidifying point of agar?

95°C and 45°C respectively.

How is chocolate agar prepared?

By adding 5% blood to hot, melted nutrient agar. It is also known as lysed blood agar.

Name the serum containing media.

Loeffler’s serum slope and Hiss serum water.

Name the egg containing media.

Dorset egg medium and LJ medium.

Name the media, which enhances Staphylococcal pigment production.

Milk agar and glycerol monoacetate agar.

What is the melting point of gelatin?

24°C. It remains as liquid when incubated at 37°C.

Which medium is used to selectively grow gram positive bacteria?

Colistin-Nalidixic acid Agar (CNA).

Name some lactose fermenters and non-lactose fermenters.

• Lactose fermenters are Klebsiella pneumoniae and Escherichia coli
• Non-lactose fermenters are Salmonella typhi, Proteus spp, Shigella spp, Pseudomonas aeruginosa, Vibrio cholerae

Name the media used for cultivating anaerobes.

Robertson’s Cooked Meat medium (R.C.M), chopped medat glucose broth and Thioglycollate broth are the liquid media. Blood agar with suitable blood agar base, reducing agents and nutrition supplements can be used to grow anaerobes.

What are the media used for blood culture?

Glucose broth, Bile broth and Brain heart infusion broth.

Why is agar preferred to gelatin?

Agar is required in smaller quantity (2-3%, in contrast to 10-15% of gelatin), is solid at the incubating temperature (while gelatin is liquid at this temperature) and does not contribute to the nutritional property of the medium (gelatin is liquefied by some bacteria).

What are indicator media?

Indicator media indicates the presence of certain bacteria by the change in colour. MacConkey's medium indicates the growth of lactose fermenters by pink coloured colonies.

What are differential media?

Media that aid in differentiation among bacteria based on some phenotypic property (such as sugar fermentation) are differential media. MacConkey differentiates LF from NLF.

What do you mean by enriched media?

Media that are supplemented with extra nutrition in the form of blood, serum, egg yolk etc are enriched media. They are suitable for fastidious bacteria.

What is biphasic medium?

Biphasic medium contains both solid and liquid medium in the same bottle. Blood is inoculated into the liquid medium and subcultures are performed by tilting the bottle. Used in Castaneda method of Blood culture.

FAQ in bacterial genetics

What is the nature of bacterial chromosome?

Bacterial chromosomes are circular and supercoiled. Borrelia burgdorferi and Streptomyces have linear chromosomes. Bacteria have single set of chromosomes, ie. they are haploid.

What is mutation?

Sudden heritable change in a nucleotide sequence. They are classified as base substitution, frame shift mutation and insertional inactivation.

What is mutatiion rate?

Mutation rate is the estimation of the rate (per generation) of mutation per nucleotide, per locus, or the whole genome. With respect to antibiotic resistance, it is frequently defined as the frequency at which detectable mutants arise in a bacterial population in the presence of a given antibiotic concentration in vitro. Generally, the mutation rate in bacteria is approximately 0.003 mutations per genome per cell generation.

What is base substitution mutation?

When one base is inserted in place of another, it is base substitution, when this results in a codon that causes a wrong amino acid to be inserted, it is missense mutation and when a base substitution results in the generation of a termination codon, it is nonsense mutation.

What are the methods of gene transfer between bacteria?

Transformation, conjugation and transduction.

What are the transposons or “jumping genes”?

These are pieces of DNA that move from one site to the another, either within or between the DNA of bacteria, plasmids and bacteriophage.

What is the significance of Transposons?

• They can code for drug resistance, enzymes and toxins.
• They can cause mutation in genes, where they insert. Such mutations are called insertional mutagenesis.

What are insertion-sequences?

Insertion sequences are short stretches of DNA that can move from one part of chromosome to another. They differ from transposons in that they code only for genes responsible for self-transfer. These may bring about insertional mutations by inactivating a gene or block the expression of gene by inserting insterting into promoter regions of operons.

What are integrons?

Integrons are mobile genetic elements that are able to capture and incorporate gene cassettes by site-specific recombination. The gene cassettes may contain genes for antibiotic resistance.

What are gene cassettes?

Gene cassette are a type of mobile genetic elements, which contain a gene and a recombination site. The cassettes may exist inside an integron or freely as circular DNA. Often, the gene cassettes carry antibiotic resistance genes.

What is transduction?

It is the transfer of DNA from one bacterium to the another by a bacteriophage.

What is conjugation?

It is the process of genetic transfer where a bacterium with F plasmid (F+) mates with another bacterium (F-) and sends a strand of F plasmid DNA into the recipient cell through sex pili.

What is F factor?

The transfer factor, also called fertility factor is a plasmid that codes for the synthesis of sex pili and self transfer.

What is R factor?

It is a plasmid that codes for drug resistance. Its transfer to other bacteria is independent of F factor.

What is an episome?

It is a plasmid, which is known to integrate itself with the bacterial chromosome.

What is the significance of plasmids?

They code for the following properties:

• Antibiotic resistance.
• Resistance to heavy metals, such as Hg, Ag.
• Resistance to UV light, which is mediated by DNA repair enzymes.
• Pili, exotoxins and bacteriocines.

What is sexduction?

The F factor is usually an episome that integrates itself into the bacterial chromosome. When such episome is transferred to F- cells during conjugation, some of the host chromosomal genes are also transferred.

FAQs in antibiotics

What is an antibiotic?

Molecules of biological origin, which are derived from soil-fungi or bacteria that are inhibitory or lethal against other bacteria are termed antibiotics. Many antimicrobial drugs are either modification of natural molecules or synthetically made.

Who coined the term "antibiotic"?

Selman Abraham Waksman, who also discovered Streptomycin, coined the term antibiotic.

Do all antibiotics kill bacteria?

Some antibiotics are bactericidal whereas some other are bacteriostatic.

What are the different mechanisms of action of antibiotics?

• Inhibition of cell wall synthesis.
• Alteration of cell membrane function.
• Inhibition of protein synthesis.
• Inhibition of nucleic acid synthesis.

Name the antibiotics that inhibit cell wall synthesis.

beta-lactam antibiotics (such as Penicillins and cephalosporins), vancomycin, bacitracin and cycloserine.

Name the antibiotics that alter cell membrane function.

Colistin, Polymyxin, Amphotericin B and Nystatin.

Name the antibiotics that inhibit protein synthesis.

Chloramphenicol, Erythromycin, Clindamycin, Tetracycline and Aminoglycosides.

Name the antibiotics that inhibit nucleic acid synthesis.

Sulphonamide, Trimethoprim, Quinolones and Rifampin.

What are the different methods of drug resistance in bacteria?

• Production of enzymes that inactivate the drug. Eg: β-lactamase, Chloramphenicol transacetylase.
• Production of new or modified targets with little or no affinity for the drug. Eg: Resistance to penicillin by MRSA, resistance to Streptomycin and Erythromycin.
• Decreased membrane permeability and active efflux. Eg: Resistance to Tetracycline.

What is the genetic basis of drug resistance?

• Chromosomal mutations.
• Acquisition of plasmid or transposon, containing gene coding for drug resistance.

What is intrinsic and acquired resistance?

• A bacterium found resistant to an antibiotic since the very beginning is intrinsically resistant to that antibiotic. Eg: Novobiocin resistance in S.saprophyticus.
• A bacterium acquiring resistance to an antibiotic through plasmid, transposon or due to a chromosomal mutation is acquired resistance. Eg: Penicillin resistance.

What are the methods of antibiotic susceptibility testing?

• Micro-broth and Macro-broth dilution method.
• Agar dilution method.
• Disk diffusion method.
• E test
• Automated systems

What are MIC & MBC?

• MIC is minimum inhibitory concentration. It is the highest dilution of an antibiotic that inhibits the growth of bacteria.
• MBC is minimum bactericidal concentration. It is the highest dilution of an antibiotic that kills the bacteria.

Name two drugs, when given in combination, are additive or synergistic.

Penicillin and Gentamicin; Amoxycillin and Clavulanic acid; Trimethoprim and sulphamethoxazole. Several drugs are given together in the treatment of tuberculosis to achieve synergistic killing and to prevent the emergence of drug resistance against any one of them.

Name two drugs, when given in combination, are antagonistic.

Penicillin and Tetracycline (since one is bactericidal and the other is bacteriostatic).

Name some bactericidal and bacteriostatic drugs.

Penicillins, Cephalosporins, Aminoglycosides etc., are bactericidal while Chloramphenicol and Tetracycline are bacteriostatic.

Which is the enzyme that degrades penicillin ring?

β-lactamase ( penicillinase and cephalosporinase). There are many types of beta-lactamases such as Extended Spectrum Beta-Lactamases, AmpC beta-lactamases, Oxacillinases and Carbapenemases.

When is a bacterium called “Multi- drug Resistant”?

When a bacterium develops acquired resistance to three or more standard antibiotics, it is said to have developed multi drug resistance. This feature is common in Staphylococcus aureus, Salmonella typhi and Mycobacterium tuberculosis. There are organisms which exhibit extended-drug resistance and pan-drug resistance.

Name some Beta-lactamase inhibitors.

Natural or derived molecules such as Clavulanic acid, Sulbactam and Tazobactam. Avibactam is a synthetic beta-lactamase inhibitor.

Name antibiotics used for topical application?

Bacitracin, Neomycin and Mupirocin.

How is the antibiotic susceptibility test reported?

Bacteria may be susceptible (sensitive), moderately (intermediate) susceptible or resistant to an antibiomicrobial drug.