Bacteriologist

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.

The cell walls of Mycobacterla are made up of waxy substance, Mycolic acid that Is relatively Impermeable to ordinary stainIng techniques. But, by apphcation 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.

Phenol. Lysol, Formaldehyde. Sodium hypochlorite.

☛ Tincture Iodine - 2% of Iodine in 70% alcohol - lodophore - Povidone Iodine.

☛ Name some antiseptics.

☛ Chlorhexidine. Chloroxylenol. spirit (70% alcohol), tincture of Iodine. H202.

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.

Sludge particles which are produced in raw or settled wastewater, by the growth of organisms in aeration tanks. This is all done in the presence of dissolved oxygen. This sludge contains living organisms that can feed on incoming wastewater

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.

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.

Sputum smears for Mycobacterla can be stained by fluorescent dyes such as Auramine and Rhodamlne as they have affinity for mycolic acid In their cell walIs 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.

Alkylation (hydrogen atom is replaced with an alkyl group) of protein. DNA. and RNA afFects bacterial metabolism and replication. EQ gas (8.5%) is often mixed with stabilizers such as CO2 (91 5%) or hydrochlorofluorocarbons (HCFC). This requires high humidity (40-80%) and long exposure times (1-6 hrs).

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.

The process of kdling all hying forms including spores is called sterilization and the process of killing of only the vegetative form of pathogenic bacteria as well as other microbes is disinfection

Beaded appearance is used to describe the appearance of Mycobacterla 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.

An organized system of meticulously constructed narrow trenches, which are partially filled with washed gravel or crushed stone, into which a pipe is placed. Discharges from septic tanks are passed through these trenches.

The cytoplasm of Gram positive bacteria are said to be more acidic

than those of Gram negative ones.

Hence the dye is said to bind with more affinity to Gram positive cells.

Primary stain: Strong Carbol Fuchsin (contain Basic fuchsin and Phenol)

Decolourizer 20% sulphuric acid

Counterstain LoelTler’s Methylene blue or 1% Malachite green, Picric acid for color-blind workers