microbiological Techniques [STM 312] CLASS FOUR

microbiological Techniques [STM 312] CLASS FOUR

GRAM STAINING PRINCIPLE

Crystal violet combine to form a large molecule which precipitate within the cell. The gram reaction is dependent on permeability or the bacteria cell wall and cytoplasmic membrane to the crystal violet iodine complex.

      Alcohol causes dehydration of the peptidoglycan layer of the cell wall. This causes decreasing of the space and the complex is trapped in and therefore therefore not lost. The primary dye is therefore retain and not decolourized. Gram positive bacteria, because of their acidic protoplasm therefore bind more firmly to the basic dye. Gram negative bacteria on the other hand have an outer membrane of phospholipid and lipoploysaccharides, outside the thin peptidoglycan layer.

   During the colorization stage alcohol dissolve this liquid as well as the membrane to which the peptidoglycan is attached, therefore the crystal violet iodine complex initially form is not retained and it wash out of the cell. When the counter stain is added, they take up the pink colour from the stain and therefore appear pink/pinkish under the microscope.

Common errors that you can make during gram staining

1.  Excessive heat use during fixation
2. The use of the low concentration of crystal violet
3. Excessive washing between the steps
4. insufficient iodine exposure
5. prolonged decolourization
6. Excessive counter staining.

Acid-fast stain

         Another  differential staining technique is known as the acid fast staining technique other wise known as ziehl Neilson. This technique differentiate species of micro bacterium tuberculosis from other bacteria. Heat or a liquid solvent is first of all used to carry the first stain carbolfushin (pinkish red in colour) Other bacteria will lose the stain when the cells are wash with a dilute alcohol solution.

                The micro-bacterium will resist the effect of the acid alcohol and retain the colour of carbolfushin stain appearing bright red. Other bacteria will lose the stain and take up the new stain of methlyene blue. 

                Microbacterium tuberculosis resist the decolouration of acid alcohol which confers the properties of acid fastness to it (resistance to decolourization by acid).

               Principle of acid fast staining

The acid fasteness of micro bacterium (tuberculosis) resist the decolourization of acid alcohol and this confer the property of acid fasteness to it.

  The acid fasteness of the acid fast bacteria is attributed to the present of large quality of wax that is present in their cell wall as well as the intactness in the cell wall.

The degree of acid fasteness varies with different bacterium. In this staining method the application of heat helps the dye to penetrate the tubercubacillus. Once it is stained the stain can not be easily removed, in non acid fast bacilli it will readily absorb the counter stain which is methylene blue and appear blue underneath the microscope and the microbacterium appears pinkish-red.

 Procedure

1. make a smear ,air dry and then heat fix
2. flood the smear with carbolfushin
3. steam for three to five minutes add more carbolfushin stain as it is required
4. wash of with distilled water
5. flood the slide with acid alcohol for a few seconds (decolorization)
6. tilt the slide over the zinc and add acid alcohol until all the red colouration stop streaming from the slide
7. rinse with distilled water and counter stain with methylene blue after which you rinse the slide again and let it dry
8. use oil immersion objective to view.

SPORE STAINING

Spores are highly resistant and metabolically inactive within the bacteria cells. A special staining technique is used to examine bacteria spores in this staining techniques, malachi-green is used with heat to force the stain into the cells and give them colour, a counterstain safranin is then used to give colour to the non-spore forming bacteria. (The spore bearing appaears green and the vegetative cell appears red).

PROCEDURE

1.     Make a smear

2.     Place the slide over a beaker of boiling water and when large droplet has condensed on the down part of the slide.

3.     Malachi green is added to the sear, leaving it for one minute once the water continues to boil, cool and wash in cold water, and counter stain with safranin red, spores appears green under the oil immersion objective, while the vegetative cells appear red.

CAPSULE STAINING

     The capsule serves as a protective material by slowing down or preventing penetration of chemicals and body juices. Chemically, the capsular material is a polysaccharide, a glycoprotein or a polypeptide capsule staining is more difficult than other types of differential staining procedures because the capsular materials are water soluble may be dislodged or removed I, rigorous washing. During capsule staining, the smear should not be heated because the resultant cell shrinkage may create a clear zone around the organism and then may be mistaken for the capsule. The capsule is non-ionic so the dyes commonly used will not bind to it.

PROCEDURE

1.     Make a smear from a colony of S.pneumoniae which is an organism that has a capsule.

2.     Do not heat fix.

3.     Flood with crystal violet and allow it to stain for 5-7 minutes.

4.     Wash the smear with 20% CuSO4 solution and hot dry.

 Capsule seen as light blue in contrast to deep colour purple of the cell.

Negative staining can also be used to observe capsules under the microscope. The procedure is as follows:

1.     Put a large loop-full of undiluted India-ink on the slide.

2.     Add a small loop-full of liquid bacterial culture to the India ink and emulsify.

3.     Take a clean grease free cover slip, and place on the ink drop and press it down so that the film becomes very thin and plane in colour.

              Under the high power objective, the capsule will be seen as a clean refractile halo around the organism against a black background.

STAINING OF SPIROCHAETES

The 3 important groups of spirochaetes are;

1.     Treponema

2.     Leptospira

3.     Borellia

       They are motile, elongated, flexible spiral organisms. They are best observed in unstained wet films under the dark microscope where they appear bright. The method of silver impregnation is usually used to thicken them which allows them to be seen.

PROCEDURE

          Threat the smear 3 times for 30 seconds each time with the fixative. Wash off the fixative with absolute alcohol and allow the alcohol to act for 3 minutes. Drain off the excess alcohol and carefully turn off the excess remainder until dry. Pour on the mordant and heat until steam rises. Allow this act to act for 30 seconds. Wash well in distilled water. Dry and mount in Canada balsam before viewing under the microscope.

          Canada balsam is used instead of immersion oil because some immersion oils cause the film to fade at once. The spirochaetes are stained brownish black on a brownish yellow background.

FLAGELLA STAIN

          Flagella is the organ for locomotion which is one or more unbranched long filament presence or absence of flagella and their number and arrangement are characteristic of different general of bacteria. Flagella are visualized by the special staining techniques in which their thickness is increased by the addition of a mordant.

PROCEDURE

          Grow the bacteria for 16-24 hours on non-inhibitory medium e.g. trypticase soy agar or blood agar. Touch a loop-full of water onto the edge of a colony and let motile bacteria swim into it. Then transfer this loop-full of water containing the motile bacteria onto a slide to get a rainthy turbid suspension and cover with a cover slip.

          The bacterial suspension is then prepared with a minimum of agitation which would detach the flagella. After 5-10 minutes when many bacteria may have attached to the surface of the slide and the cover slip, apply 2 drops of Ryus stain to the edge of the cover slip and leave the stain to diffuse into the film. Examine with the microscope after standing 5-15 minutes at ambient temperature.

FOR FUNGI

          Lactophenol cotton blue (LPCB) is used to demonstrate fungi and fugal elements. On a clean glass slide, place a drop of LPCB touch the adhesive side of a transparent cello-tape on the surface of the colony and fix the adhesive side of the tape over an area on the glass slide containing LPCB. Examine under the microscope after 30 minutes to give sufficient time for the structure to take up the stain.