- EXPERIMENT 11 TECHNIQUES Structure 11.0 Objectives 11.1 Introduction 11.2 Preparation of Smear 11.3 Gram Staining Technique 11.4 Ziehl-neelsen Staining Technique 11.5 StainingTechnique (Loeffler) 11.6 Giemsa Staining Technique 11.7 Hiss Staining Technique 11.8 Lactophenol Cotton Blue (LPCB) Staining 11.9 Precautions 11.10 Activity 11.0 OBJECTIVES

After going through this experiment you will be able to:

prepare a smear for the study of microorganisms;

stain the smears for identification of the microbes; and

explain different staining methods and their utility.

11.1 INTRODUCTION

Staining of the clinical material or the from colonies on laboratory media provide a direct visualization of the morphology of the organisnls as well as their reactions to the chemicals present in stains. This is an invaluable and easy-to-use tool for establishing the identity of various microorganisms. Some of the commonly-used staining techniques are described here. 11.2 PREPARATION OF SMEAR

To prepare bacteria for staining a sample of bacteria in liquid is spread as a thin layer or smear on a clean glass slide. The thin bacterial film is allowed to air dry and then it is fixed to the glass to prevent it from washing off during the subsequent staining procedure, by passing the slide briefly through a flame; this treatment sticks (glues) the bacteria to the glass and also kills any pathogens. The bacterial film should not be too thin or too thick. Once you have a fixed bacterial film it is then stained. Mark a circle on the under side of a grease free slide with a glass etching tool. Several circles can be located on the same slide. Sterilize the inoculation needle in the flame and allow it to cool down and place a Practical Manual - very small drop of distilled water over the circled area in case of a dry culture. Fundamentals of Food and Meat Science After aseptically removing material from a culture, mix it with the drop or placed directly on the slide if it is a dilute broth culture. It takes very little material to produce a successful smear. Air-dry the smear and then fix it by quickly passing the slide through a flame. Three quick passes are usually sufficient to kill the bacteria and cause them to adhere. After cooling the slide, .the smear is ready for staining procedure.

11.3 GRAM STAINING TECHNIQUE

This is the most extensively used technique to differentiate most bacteria by their Gram reaction due to differences in their wall structure. Those organisms are called Gram- positive which after being stained dark purple or bluish purple with are not decolourized by acetone or ethanol. Those organisms are called Gram-negative which after being stained with crystal violet lose their colour when treated with acetone or ethanol, and stain pinklred with , , or other red counter stain. The difference of colour retaining property is because of difference in nature of cell membrane. Gram-negative bacteria have high lipid content in their cell walls. Decolourizer (acetone or ethanol) solubilizes the outer membrane of the cell wall thus releasing the crystal violet. In the cell wall of Gram-positive organism, the decolourizer is unable to act as a solvent thus the crystal vi~letremains. The iodine solution used in the technique acts as a mordent for crystal violet. The Gram reactions of some important bacteria are as follows.

Gram-positive : Staphylococcus, Streptococcus, Clostridium and Corynebacterium. Gram-negative : Neisseria, Klebsiella, Haemophilus, Coliforms, Yersinia, Shigella and Vibrio. Requirements 1) Crystal violet stain 2) Lugol's iodine 3) Acetone-alcohol decolourizer 4) Safranin solution Ingredients and preparation Crystal Violet Sohtwn Solution A Crystal violet 2.0 gm Ethanol, 95% 20 ml Solution B Ammonium oxalate 0.8 gm Distilled water 80 ml Mix solutions A and B. Store for 24 hours before use. a Gram Iodine (Lugol's iodine) Solution Staining Techniques Iodine crystals 1.0 gm Potassium iodide 2.0 gm Distilled water 100 ml Grind the dry iodine and potassium iodide in a mortar. Add water, a few rnl at a time, and grind thoroughly after each addition until the iodine and iodide dissolve. Rinse the solution into an amber glass bottle with the remainder of the distilled water. a Safranin Solution Stock solution Safranin 0 2.5 gm Ethanol, 95% 100 ml Working solution Stock solution 10 ml Distilled water 90 ml Method

1) Fix the dried smear by rapidly passing the slide, smear uppermost, three times through the flame of spirit lamp. 2) Cover the fixed smear with crystal violet stain for 30-60 seconds.

3) Rapidly wash off the stain with clean water. 4) Tip off all the water, and cover the smear with Lugol's iodine for 30-60 seconds. 5) Wash off the iodine with clean water. 6) Decolourize rapidly (few seconds) with acetone-alcohol. Wash immediately with clean water.

7) Cover the smear with Safranin solution for 30 seconds. 8) Wash off the stain with clean water 9) Wipe the back of the slide clean, and place in a draining rack for the smear to air- dry. 10) Examine the smear microscopically, first with the 40X objective to check the staining and to see the distribution of material, and then with the oil immersion objective to look for bacteria and cells. Open fully the condenser iris when using the oil immersion lens. Results Gram positive bacteria ...... Dark purple Yeast cells ...... Dark purple Gram negative bacteria ...... Red Practical Manual - Fundamentals of Food 11.4 ZIEHL-NEELSEN STAINING TECHNIQUE and Meat Science The Ziehl-Neelsen (ZN) technique is used to stain Mjlcobacterium species including M. tuberculosis, M. ulcerans, and M. leprae. Mycobacteria, unlike most other bacteria, do not stain well by the Gram technique. Mycobacteria can be stained with ~arbolfuchsin, which consists of a strong basic dye called basic fuchsin combined with phenol. After staining, an acid decolourizing solution is applied which removes the red dye from the background cells, tissue fibres, and any organisms in the smear except those that are acid fast. The bacilli that retain (hold fast to) the are referred to as Acid Fast Bacilli. The difference is because of presence of thick, waxy coat in Mycobacteria. Non acid fast bacteria are decolourized and take up the counter stain. Following decolourization, the smear is counterstained with malachite green or methylene blue which stains the background material and provides a good contrast against which the red Acid Fast Bacilli can be seen. Some actinomycetes, corynebacteria, and bacterial endospores are also acid fast. Requirements

1. Carbol fuchsin 1%

2. Sulphuric acid 25%

3. Methylene blue 0.1% ' Method

1) Alcohol-fix the dried smear by covering it with one or two drops of 70% vlv ethanol or methanol for 2-3 minutes. It has been shown that heat- does not kill M. tuberculosis in sputum smears. The organisms are killed, however, by alcohol fixation, and this method should be used routinely especially when smears are not stained immediately.

2) Cover the smear with the filtered carbol fuchsin stain heat the stain until vapour just begins to rise (i.e. about 60°C). Do not overheat.

3) Allow the heated stain to remain on the slide for 5 minutes.

4) Wash off the stain with clean water. 5) Cover the smear with 3% vlv acid alcohol for 5 minutes or until the smear is sufficiently decolourized, i.e. pale pink. Acid alcohol is flammable, therefore use with care well away from an open flame.

6) Wash well with clean water. 7) Cover the smear with malachite green stain for 1-2 minutes, using the longer time if the smear is thin.

8) Wash off the stain with clean water. 9) Wipe the back ofthe slide clean, and place in a draining rack for the smear to air- dry (do not blot dry).

10) Examine the smear microscopically first with the 40 X objective to see the distribution of material and then systematically with the oil immersion objective to look for acid- fast bacteria. Open fully the condenser iris when using the oil immersion lens. I Result Staining Techniques Acid Fast Bacilli (AFB) ...... Red, straight or slightly curved rods, occumng singly or in small groups. Cells ...... Green Background material ...... Green. 11.5 METHYLENE BLUE STAINING TECHNIQUE (LOEFFLER)

This rapid method can be used to show the basic morphology of bacteria. It is also useful for staining leucocytes in faecal preparations. The stain is used to make out clearly the morphology of the organisms e.g. Yersinia pestis in exudate, Haemophilus influenzae in CSF and gonococci in urethral pus. The cells contains volutin gramules which consist of polymetaphosphate. Polymetaphosphate have a strong affinity for basic methylene blue dye, the granules usually stain metachromotically and appear pink-red. The cells stain pale blue. Requirements Loeffler methylene blue or Polychrome Loeffler methylene blue. Ingredients and preparation Methylene blue -4.3 gm Distilled water -100 rnl Dissolve the dye in u ilter. Filter through a filter paper. Method

1) Fix the dried smear. If anthrax is suspected, fix the smear with potassium pennanganate for 10 minutes.

2) Cover the smear with the stain for 1 minute. 3) If staining anthrax bacilli, use Loeffler's polychrome methylene blue. 4) Wash off with clean water. 5) Wipe the back of the slide clean, and place in a draining rack for the smear to air- dry. 6) Examine the smear microscopically, first with the 40 X to see the distribution of material, and then with the oil immersion objective to look for bacteria. Results

Bacterial cells --- -- Blue

Nuclei of leucocytes ---- Blue

Capsular malerial - Mauve-purple Practical Manual - Fundamentals of Food 11.6 GIEMSA STAINING TECHNIQUE- and Meat Science Giemsa is a that is widely used in parasitology to stain and other blood parasites. The stain contains azure B and Y and is capable of making subtle distinction in the shades of staining. The acidic groupings of the nucleic acids and proteins of the cell nuctei determine their uptake of the basic dye azure B and the presence of basic groupings result in an affinity for acidic dyes & their staining by eosin. In microbiology, the Giemsa technique is used mainly to stain Chlamydia trachomatis inclusion bodies, Borrelia species, and if Wayson's stain is not available, to stain Yersinia pestis. Requirements Buffered water, pH 7.0-7.2 Method

1) Fix the dried smear by covering it with methanol for 2-3 minutes. Allow the smear to air-dry.

2) Dilute the Giemsa stain as follows: Dilute the stain 1 in 20. i. Fill a small cylinder to the 19 ml mark with the buffered water. ii Add 1 ml of Giemsa stain, i.e. to the 20 ml mark.

3) Place the slide smear downwards, in a petridish or other small container, supported on each side by a thin piece of stick.

5) Pour the diluted stain into the dish and cover with a lid. 5) Leave the smear to stain for 25-30 minutes. 6) Wash the stain from the dish and rinse the smear with buffered water. 7) Wipe the back of the slide clean, and place in a draining rack for the smear to air- dry. 8) Examine the smear microscopically, first with the 40X objective to see the distribution of material and to select a suitable part of the smear to examine with the oil immersion lens. Results granules ...... Red Melanin granules ...... Black-green Bacteria ...... Pale or dark blue Borrelia spirochaetes ...... Mauve-purple Red cells ...... Mauve-pink Nuclei of white cells ...... Dark purple Cytoplasm of white cells ...... Pale blue or grey-blue Staining Techniques 11.7 HISS STAINING TECHNIQUE

The Hiss technique is used for staining the capsules around bacteria. The polysaccharide or polypeptide composition of the capsule makes staining difficult. Capsule appears as pale blue halos around deep blue to purple cells. Requirements Crystal violet, 10 g/l(l% wlv) stain Copper sulphate, 200 g1I (20% wlv) Method

1) Fix the dried smear using alcohol. 2) Cover the smear with crystal violet stain, and heat gently until the steam just begins to rise. Leave to stain for 1 minute. . 3) Wash off the stain with the copper sulphate solution. 4) Wipe the back of the slide clean, and place in a draining rack for the smear to air- dry.

5) ExanLine the smear microscopically, first with the 40 X objective to see the distribution of material and then with the 100 X oil immersion objective to look for capsulated bacteria. Results Bacterial cell Dark purple Capsule .outline Pale blue. 11.8 LACTOPHENOL COTTON BLUE (LPCB) STAINING

The LPCB stain is formulated with lactophenol, which serves as a mounting fluid, and cotton blue. Cotton blue is an acid dye that stains the chitin present in the cell walls of fungi. Organisms suspended in the stain are killed due to the presence of phenol. The high concentration of the phenol deactivates lytic cellular enzymes thus the cells do not lyse. Lactic acid preserves the fungal structures. Requirements Phenol 200.0gm Cotton Blue 0.5gm Glycerol 400ml Lactic Acid 200ml Deionized Water 200ml The final volume is made upto one liter by distilled water. Preparation of LPCB slide mounts LPCB wet mount preparation is the most widely used method of staining and observing fungi and is simple to prepare. It can be used to also look at filaments and higher life Practical Manual - forms with microsco~icwork. Just remember. the stain will slow down and or cause the Fundamentals of Food higher life forms to die. Use a normal wet mount first for higher life form counts. and Meat Science Procedure for Staining: . Place a drop of wastewater sample on a microscope slide. Add one, or at most two drops of the Lactophenol Cotton Blue stain. Folding the coverslip between forefinger and thumb, touch one edge of the drop of sample with the coverslip edge, and lower gently, avoiding air bubbles. The preparation is now ready for examination. If desired, seal the edges of the coverslip with nail polish or permount to preserve the mount as a reference slide. 11.9 PRECAUTIONS

Always take a clean, dry, non greasy slide for smear preparation. For fixation of slide never heat it much. Marking of slide should be done on comer to recognize the sides. Its better to keep the slides on slide stand instead of keeping on surface. The gap between two stain, decolourizer and washing should be proper. Always prefer to use fresh solution. 11.10 ACTIVITY

AfCer going through this experiment prepare two slides from different culture and perform Gram staining and ZN staining.