Gram Staining Techniques in Microbiology, Slides of Microbiology

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STAINING TECHNIQUES

IN MICROBIOLOGY

Microbiology and Immunology Lab

INTRODUCTION

 As bacteria consist of clear protoplasmic

matter, differing but slightly in refractive index

from the medium in which they are growing, it

is difficult with the ordinary microscope,

except when special methods of illumination

are used, to set them in the unstained

condition.

 Staining, therefore, is of primary importance

for the recognition of bacteria.

Staining may be simple staining and

differential staining.

STAINING METHODS

 (^) POSITIVE STAINING: - where the actual cells are themselves colored and appear in a clear background. (a) Simple staining: A stain which provides color contrast but gives same color to all bacteria and cells. Ex: Loeffler’s methylene blue, Polychrome methylene blue, Diluted carbol fuchsin. (b) Differential Staining: A stain which imparts different colors to different bacteria is called differential stain(which contains more than one stain). Ex: Gram’s stain , Acid fast staining, Special stains.  (^) NEGATIVE STAINING: where the cells remain clear (uncolored) and the background is colored to create a contrast to aid in the better visualization of the image. a) Indian ink b) Nigrosin.

BACTERIAL SMEAR

PREPARATION:

Smear - is a distribution of bacterial cells on a slide for the purpose of viewing them under the microscope. Method:  -Aseptically a small sample of the culture is spread over a slide surface.  -This is then allowed to air dry.  -The next step is heat fixation to help the cells adhere to the slide surface.  -The smear is now ready for staining.

SMEAR FIXATION

Heat fixation a) Pass air-dried smears through a flame two or three times. Do not overheat. b) Allow slide to cool before staining. Methanol fixation c) Place air-dried smears in a coplin jar with methanol for one minute. d) Alternatively, flood smear with methanol for 1 minute. e) Drain slides and allow to dry before staining.

GRAM STAINING

 Gram staining is most widely used differential staining in Microbiology.  Gram staining differentiates the bacteria into 2 groups:  Gram positive.  Gram negative.

ORIGINAL FORMULATION OF DR. GRAM  Aniline Gentian violet,  Lugol’s Iodine,  Absolute Alcohol,  Bismarck Brown

CARL WEIGERT (1845-

 (^) German pathologist Carl Weigert (1845-1904) from Frankfurt, added a final step of staining with safranin. In his paper, Dr. Gram described how he was able to visualize what we now call Staphylococcus, Streptococcus, Bacillus, and Clostridia in various histological sections. Interestingly, Dr. Gram did not actually use safranin as a counter stain in the original procedure (Gram negative cells would be colorless). He instead recommended using Bismarck brown as a counter stain to enable tissue cell nuclei to be visualized.

GRAM POSITIVE BACTERIA

 (^) Gram positive bacteria have a thick cell wall of peptidoglycan and other polymers. Peptidoglycan consists of interweaving filaments made up of alternating N-acetylmuramic acid and  (^) N- acetylglucosamine monomers. In Gram positive bacteria, there are "wall teichoic acids". As well, between the cell wall and cell membrane, there is a "membrane teichoic acid".

GRAM NEGATIVE BACTERIA

 (^) Gram negative bacteria have an outer membrane of phospholipids and bacterial Lipopolysaccharides outside of their thin peptidoglycan layer.  (^) The space between the outer membrane and the peptidoglycan layer is called the periplasmic space.  (^) The outer membrane protects Gram negative bacteria against penicillin and lysozymes

METHOD CONSISTS OF

FOUR COMPONENTS:

 Primary stain—Crystal violet, Methyl violet & Gentian violet.  Mordant—Gram Iodine, Rarely Lugol’s Iodine.  Decolourizer—Alcohol,Acetone, Alcohol: Acetone (1:1) mixture.  Counter stain—Dilute Carbol fuchsin, Safranin, Neutral red, Sandi ford stain for Gonococci

RESULT

 (^) Bacteria that manage to keep the original purple dye have only got a cell wall - they are called Gram positive  (^) Bacteria that lose the original purple dye and can therefore take up the second red dye have got both a cell wall and a cell membrane - they are called Gram negative.