Microbiology Lab (3)Differential Stains (Gram stain & Acid Fast Stain)

Abdelraheem BA

To learn how to stain bacteria with gram stain & acid-fast stain.

To determine the form, gram reaction, shape and arrangement of stained bacteria.

Objectives

A stain that is used to differentiate between certain groups of bacteria.

This techniques requires the use of:◦ At least 3 chemical reagents.◦ Heat fixation of prepared bacterial smears.

Examples:◦ Gram stain: Gram +ve Vs Gram –ve bacteria.◦ Acid-fast stain: Acid fast Vs Non-Acid fast.

Differential stains

A technique that is used to stain bacteria and yeast.

One of the most commonly performed procedures in microbiology lab.

A very important step in the identification of bacteria.

Gram stain

Primary stain: Crystal violet. Mordant*: Gram’s Iodine. Decolorizing agent: 95% Alcohol or

acetone-alcohol (50:50). Counter stain: Safranin

*Mordant:◦ A substance that forms a colored insoluble complex

by binding the primary.◦ CV-I complex hardly leave the cell.◦ Result: intensified color & fixation of stain.

Reagents in Gram stain

Allow the smear to completely air dry. Heat fixation. Place the slide on the staining tray. Flood the smear with the primary stain

(Crystal violet) for 1 minute. Wash the smear with tap water to remove

extra stain. Flood the smear with iodine for 1 minute. Wash the smear with tap water to remove

extra iodine.

Steps to perform gram stain

Rinse with the decolorizing agent (a crucial and a sensitive step);◦ Few drops for 10 seconds.

Immediately wash the slide with tap water, to remove extra stain.

Flood the smear with the counter stain (Safranin) for 1 minute.

Wash with tap water. Use bibulous paper to dry the stained smear

and examine with light microscope.

Steps – Cont’d

Bacterial cell wall review:◦ G+ve:

Thick layer of peptidoglycan. Thin layer of lipids.

Lipids will be dissolved by alcohol (forming minute pores). These pores will then shrink by the dehydrating effect of

alcohol. CV-I complex will be prevented from leaving.

◦ G-ve: Thin layer of peptidoglycan. Thick layer of lipids.

Due to alcohol, lipids will dissolve forming large pores. CV-I complex will leave, the bacteria become colorless.

What is the principle?

Bacterial cell wall

Gram Stain - Summary

Decolorization is a crucial step;◦ Over decolorization; loss of primary stain (False gram –ve

bacteria)◦ Insufficient decolorization; failure to remove CV-I complex

(Fals gram +ve bacteria). Fresh cultures must be used;

◦ Old cultures (in case of gram +ve bacteria); damage to cell wall which leads to failure to retain the CV-I complex.

◦ Gram variable result. Suitable fixation must be used;

◦ Too much heat will destroy cell wall; Gram +ve bacteria will lose the primary stain (False gram –ve

bacteria). Freshness of reagents.

Precautions

Most bacteria are stained by gram stain. Members of genus Mycobacterium and

Nocardia have a special cell wall.◦ Thick waxy layer (Lipoidal).◦ This makes these bacteria resistant to many

antiseptics and antibiotics.◦ This is the reason for being ACID FAST.

Acid Fast stain

Cell wall is composed of:◦ Fatty acids (mycolic acid).◦ Complex waxes.◦ Complex unique glycolipids.

Examples:◦ M. tuberculosis; the causative agent of TB.◦ M. leprae; the causative agent of leprosy.

Mycobacteria

Membrane of Mycobacteria

The cell wall keeps the color red of Carbol fuchsin (the primary stain), when decolorized with acid-alcohol.

Other bacteria will be decolorized and stain blue, the color of Methylene blue (the counter stain).

What does “Acid Fast” mean?

Primary Stain.◦ Carbol fuchsin; a phenolic dye and soluble in the waxy cell wall.◦ Used with the aid of heating (figure).◦ After this step, all bacteria will stain red.

Decolorizer.◦ Acid-alcohol.◦ Before addition, the slide must be cooled to harden the waxy cell

wall.◦ After this step.

Acid fast bacteria will remain red. Non-Acid fast bacteria will be colorless.

Counter stain.◦ Methylene blue.

Acid fast bacteria remain red. Non-Acid fast bacteria will stain blue.

Reagents of Ziehl-Neelsen Method

Good Luck