Staining Even with the microscope, bacteria are difficult to see unless they are treated in a way that increases contrast between the organisms and their background. The most common method to increase contrast is to stain part or all of the microbe. Preparing heat fixed bacterial smear:- A heat fixed smear is a thin layer of the bacterial specimen dried and fixed onto the slide. Procedure: 1- A circle should be marked on the under side of a slide with a glass marker. Several circles can be located on the same slide. 2- To prepare a smear from a suspended culture, aseptically transfer 3-4 loopfuls of the culture (after shaking), place directly on the slide and spread. To prepare a smear from a dry culture, a very small drop of distilled water should be placed over the circled area. After aseptically removing material from a culture it is then mixed with the drop of water. 3- Air dry or dry over the flame. 4- Heat fixation: by passing the slide three quick passes through the flame. Heat fixation:- Heat fixation accomplishes three things: (1) it kills the organisms; (2) it causes the organisms to adhere to the slide; and (3) it alters the organisms so that they more readily accept stains (dyes). If the slide is not completely dry when you pass it through the flame, the organism will be boiled and destroyed. If you heat-fix too little, the organism may not stick and will wash off the slide in subsequent steps. If you heat-fix too much, the organisms may be incinerated, and you will see distorted cells and cellular remains. - 1 -
Figure 1(Smear preparation) - 2 -
Types of staining methods: Simple stain: Is composed of one type of stain e.g, crystal violate, safranine. methylene blue (will reveal the metachromatic granules of Corynebacterium diphtheriae, the morphology of fusiform bacteria and of spirochetes in oral specimens), basic fuchsine. Differential stain: It gives different color to different bacteria e.g. Gram stain, Albert's stain, and Ziehl neelsen stain. Special stain: Negative: This procedure involves staining the background with an acidic dye, leaving the cells contrastingly colorless. The black dye nigrosin is commonly used. This method is used for those cells or structures difficult to stain directly (Figure 2). Figure 2: negative staining procedure - 3 -
India ink stain for capsule staining: Involves treatment with hot crystal violate solution followed by a rinsing with copper sulfate solution. The latter is used to remove excess stain because the conventional washing with water would dissolve the capsule. The copper salt also gives color to the background, with the result that the cell and background appear dark blue and the capsule a much paler blue. Gram stain: First devised by Hans Christian Gram during the late nineteenth century, the gram stain can be used to divide most bacterial species effectively into tow large groups: those that take up the basic dye, Crystal violet (gram positive), and those that allow the crystal violate dye to wash out easily with the decolorizer alcohol or acetone (gram negative). Staining Procedure. Materials:- Slides with heat fixed smears. gram staining kit and washing bottle. 1- Fix smear by heat. 2- Cover with crystal violet. 3- Wash with water. Do not blot. 4- Cover the smear with lugol's Iodine solution for one minute. 5- decolorize for 10-30 seconds with gentle agitation with 95% ethyl alcohol. 6- Wash with water. Do not blot. 7- Cover the smear with safranin for 10-30 seconds. 8- Wash gently with water for a few seconds, air dry, or blot with absorbent paper. 9- Examine the slide under oil immersion (Figure 3). Results: Organisms that retain the violet-iodine complexes after washing in ethanol stain purple and are termed Gram- positive, those that lose this complex stain red from safranin counter stain are termed Gram negative. Most cell wall contain Peptidoglycan, a molecule of amino acids and sugar. A distinguishing factor among Gram-positive bacteria is that 90% of their cell wall is comprised of Peptidoglycan layers. The blue - violet color reaction in Gram-positive bacteria is caused by crystal-violet,iodine complex with slow decolorizing by alcohol is observed due to closing of pores running through the cell wall. Because the crystal-violet is still present in the cell, the counter stain is not incorporated, thus maintaining the cell's blue-violet color. On the other hand, Gram negative bacteria - 4 -
incorporate the counter stain rather than the primary stain, because the cell wall of G-ve bacteria is high in lipid content and low in Peptidoglycan content, so the primary crystal violet escapes from cell wall when decolorizer is added Figure 3 : Gram stain method. - 5 -
Ziehl- Neelsen stain:- Most bacteria in the genus Mycobacterium contain very high percentage lipid in their cell wall. This stain is used in the identification of the tuberculosis bacillus, Mycobacterium tuberculosis, and the leprosy organism, Mycobacterium leprae. after decolorization, methylene blue (counter stain) is add to the organisms any material that is not acid fast Thus stained slide of mixture of acid fast organisms, tissue cells and non-acid-fast bacteria will reveal blue. acid fast bacteria will reveal red rods..0 Figure 4 (Ziehl- Neelsen stain) - 6 -
Procedure:- Ziehl- Neelsen acid- fast stain 1- Fix smear by heat. Heating helps in penetration of dye(carbol fuchsin) through waxy coat surrounding the cell wall of Mycobacteria. 2- Cover with carbol fuchsin, steam gently for 5 minutes over direct flame (or for 20 minutes over a water bath). 3- Wash with water. 4- Decolorize in acid- alcohol until only a faint pink color remains. 5- Wash with water. 6- Counter stain for 10-30 seconds with methylene blue. 7- Wash with water and let dry. 8- Examine the slide under oil immersion ( Figure 4) Albert s stain :This stain was used to diagnose Corynebacterium: Procedure:- 1- A heavy smear of bacterial isolate was prepared on glass slide and allowed to dry without fixing. 2- Albert s stain was applied for (3-4) minutes then it was washed with water and Albert s iodine was applied for one minute. 3- Wash with water and let dry with blotting paper. 4- Examine the slide under oil immersion, it was examined under microsope showed bacili with numerous metachromatic granules. Results:- Metachromatic granules Bacillary body or protoplasm Bluish- black Green - 7 -
Uses:- 1- Albert staining is useful in the demonstration of metachromatic granules. 2- Presence of metachromatic granules helps to distinguish C.diphtheriae from diphtheriods which lack them. - 8 -
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