Staining of microorganism; Purpose
To stain the microorganism in order to identify the bacteria or fungi.
Scope
This SOP is applicable to stain bacteria and Yeast/mould in Microbiology Section of General Building at XX Pharmaceuticals Limited.
Definitions
Staining
This the auxiliary technique which is mainly used to enhance contrast in microscopy on a microscopic image. The main application of stain and dyes in medicine and biology sector to highlight the structure of biological tissue.
Gram Staining
Gram stain is the most useful staining technique engaged in bacteriology, is a differential stain. By using this technique, it is possible to divide bacteria into two different groups- Gram Positive & Gram Negative.
Endospore Staining
Spore stain is a most useful staining technique engaged in bacteriology is a structural stains. By this technique, it is possible to detect the presence & position of endospore in the bacteria.
Responsibilities:
Executive/ Sr. Executive, Microbiology
Initiative of subculture of Microorganisms, perform staining & report preparation.
Manager, Microbiology
Confirm staining techniques, safety report checking, document preservation & application of sound technical information.
Head of Quality Assurance
Approval of SOP
Procedure:
Instructions
- Standard microorganisms are usually non-pathogenic but those are unscrupulous pathogen. So before handling it, wear protective items such as sterile wear sterile latex free gloves, laboratory coat & eye protection (if necessary).
- Don’t move vigorously into test area. Move always softly.
- After completion of subculture or transfer of pellets, disinfect outer surface of the vial or test or plate with 70% IPA.
- Remove all used items those are directly contacted with standard micro-organisms. Leave it at designated containers safely.
- Ensure that the waste container is tightly capped until perform autoclaving.
- Don’t touch the apparatus directly in open hands those are used.
- Make sure that all personal ornaments, cell phone are left to prevent unauthorized contamination, before entrance into the test room.
Simple Staining
Staining Reagents
Methylene blue Stock Solution :
- Methylene Blue 2.g
- Distilled Water 100 ml
Methylene blue Staining Solution :
- 0.2 % of Methylene Blue Solution 12.5 ml
- Distilled Water 87.5 ml
Crystal Violet Solution
- Crystal Violet 2.0 g
- Ethyl alcohol 95% 20 ml
- NH4 Oxalate 0.8 g
- Distilled Water 100 ml
Carbol Fuchsin(Zeihl-Neelsen) Solution
- Fuschin 1 g
- Ethanol 10 ml
- Phenol 5 g
- DistilledWater 200ml
Dissolve Fuschin in alcohol and dissolve phenol in water, then mix the two solutions.
Apparatus
- Glass slide 3 x 1 inch
- Pipettes
- Gas Burner or spirit Lamp
Staining Technique:
- Clean & dry microscope slides thoroughly.
- Flame the surface in which the smear is to be spread.
- Flame the inoculating loop properly.
- Transfer a loop full of tap Water to the flamed slide surface.
- Reflame the loop making sure that the entire length of the wire that will enter the tube has been heated to redness.
- Remove the tube cap with the fingers of the hand holding the loop.
- Flame the tube mouth.
- Touch inoculating loop to the inside of the tube to make sure it is not so hot that it will distort the bacterial cells; then pick up a pinhead size sample of the bacterial growth without digging into the agar.
- Reflame tube mouth, replace can, and put tube back in the holder.
- Disperse bacteria on the loop in the drop of water on the slide and spread the drop over an area the size of a dime. It should be a thin, even smear.
- Reflame the inoculating loop to redness including the entire length that entered the tube.
- Allow the smear to dry thoroughly.
- Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times.
- Test the temperature of slide after each pass against back of the hand. It has been heated adequately when it feels hot but can still be held against the skin for several seconds. Excessive heat will distort the cells.
- Stain smear by flooding it with one of the staining solutions & allowing it to remain covered with stain for time designated below.
- Methylene blue: 1 minute
- Crystal violet: 30 seconds
- Carbol Fuchsin: 20 seconds
- During staining the slide may be placed on the rack or held in the fingers.
- At the end of the designated time rinse off the excess stain with gently running tap water. Rinse thoroughly.
- Wipe the back of the slide and blot the stained surface with bibulous paper or with a paper towel.
- Place the stained smear on the microscope stage smear side up and focus the smear using the 10x objective.
- Choose an area of the smear in which the cells are well spread in a monolayer. Center the area to be studied.
- Apply oil directly to the smear, and focus the smear under oil with the 100X objective.
- Draw the cells observed.
Gram Staining Method
Stain and Reagents
Crystal Violet Solution
- Crystal Violet 2.0 g
- Ethyl alcohol 95% 20 ml
- NH4 Oxalate 0.8 g
- Distilled Water 100 ml
Gram’s Iodine
- Iodine Crystals 1.0 g
- Potassium Iodide 2.0 g
- Distilled Water 100 ml
Docolorizer :
- Acetone 50 ml
- Ethanol 96% 50 ml
Counterstain :
- Safranin 2.5 g
- Ethanol 95% 100 ml
- Distilled Water 100 ml
Apparatus :
- Glass slide 3 x 1 inch
- Pipettes
- Gas Burner or spirit Lamp
Staining Technique
- Use always young culture of (18 to 24) hours that the differentiation in cell wall structures is retained.
- Do not use old cultures due to lose Gram Positiveness.
- Make smear, and dry in air and fix by flaming.
- Stain with crystal violet for about 30 seconds.
- Rinse with the water.
- Cover smear with Gram’s iodine for about 30 seconds.
- Rinse with the water.
- Decolorize with 95% ethanol. For a thin smear, 10-20 seconds is long enough; after the proper time interval, alcohol
- Drippings from the slide are no longer colored.
- Rinse with the water.
- Counterstain with safranin solution for 20-30 seconds.
- Rinse with water and blot dry.
- Examine under the oil-immersion objectives.
Interpretation of Result :
- Gram positive bacteria retain the crystal violet dye after de-colorization and appear deep blue or purple colour.
- Gram negative bacteria are not capable of retaining the crystal violet dye after de-colorization and are counterstained red or pink by the safranin dye.
Endospore staining :
Stain and reagents :
- Malachine Green solution
- Malachite green 5 g
- Distilled Water 100 ml
Counterstain Solution
- Safranin 2.5 g
- Ethanol 95% 100 ml
- Distilled water 10 ml
Apparatus
- Glass slide 3 x 1 inch
- Pipettes
- Gas burner
Slide Cleaning :
- Clean the slide by immersion in concentrated Sulphuric Acid saturated with Potassium dichromate for several days for removal of grease from slide.
- Place drop water on the surface of the slide, if water is spread over its surface indicates the slide is cleaned properly.
Staining Technique
- Prepare smear, dry in air and fix by flaming.
- Place the slides on a staining rack.
- Cover the smear and keep saturated with malachite green 5% aqueous solution and continue heating for 5 minutes.
- Wash gently with the water.
- Counterstain with safranin for 30 seconds.
- Wash with water and blot dry.
- Examine under the oil-immersion objectives.
Interpretation of result :
The endospore stains green and the remainder of the cell (or a cell without an endospore) stains light red.
The phase microscope is effective in observing the endospore without staining where it appears as a dense white structure in the cell. If a phase microscope is available, observe the unstained endospore in cultures of Bacillus and Clostridium species.