Staining Techniques

On this page:

PREPARATION OF SMEARS

GRAM STAIN

ZIEHL-NEELSEN STAIN

FLUORESCENT ACID FAST STAIN

SPORE STAIN

ALBERT’S STAIN FOR METACHROMATIC GRANULES

WET INDIA INK PREPARATION FOR CAPSULES

INTRODUCTION


Micro-organisms, and bacteria in particular, are difficult to observe in the microscope if unstained. They have a refractive index close to that of the suspending medium which makes them difficult to see unless special techniques such as phase contrast microscopy are used. However, it is often more productive to stain the cells to improve contrast and to provide other information at the same time. For example, visualisation of cells in the phase microscope may tell you if they are rods or cocci, but a gram stain will also tell you if they are gram positive or negative as well, and this is often the first step in identification.

PREPARATION OF SMEARS

  1. Briefly pass a clean microscope slide through the flame of a Bunsen burner.
  2. Label the slide with a diamond pencil (or lead pencil if the slide has a ground glass end). Texta or marker pen will wash off during staining.
  3. Use a diamond pencil to mark a circular area on the slide about the size of a 10 cent piece.
  4. For liquids, place a loopful of liquid within the marked area, spread out and allow to dry completely.
  5. For solid cultures, place a loopful of saline or water into the marked area. Do not use too much liquid. Pick up a colony with the loop and emulsify in the liquid on the slide. Spread the liquid out and allow to dry.
  6. For a swab, rub the swab back and forth within the marked area, rolling the swab as you go. Allow to dry.
  7. When slides are dry, turn over and pass 3 times through the flame of the Bunsen burner to heat fix the material to the slide. Do not overheat as the material may incinerate.

GRAM STAIN

The Gram stain is the basic stain used in all microbiology laboratories. There are many variations to the technique but the basic procedure is the same. The order of the staining reagents is always:

  1. Crystal violet
  2. Gram’s iodine
  3. Decolourisation
  4. Counterstaining


Variations commonly occur in the type of decolourising agent used and the type of counterstain.
The critical step is decolourisation; not enough and gram negatives will appear gram positive, too much and gram positives will appear gram negative.
In the first step crystal violet is added to the smear and enters the cell walls of the bacteria. Iodine is then added which complexes with the crystal violet in the cell wall. This step occurs in all cells, resulting in purple stained cells at this stage.
Decolouriser is then added (acetone or acetone/alcohol) which removes the dye complex from the cell walls of gram negative cells, resulting in unstained cells. Gram positive cells are unchanged and remain purple. It is believed that the solvents used as decolourisers open up “pores” in gram negative cell walls, allowing the dye complex to be washed out. The counterstain is then applied which stains the gram negative cells red/pink. Gram positives are unaffected by the counterstain as they are already stained by the darker purple of the crystal violet complex.

PROCEDURE

  1. Place the slides smear side up on the staining rack over the sink and have the tap running gently. Flood the slide with crystal violet i.e. cover the whole smear. Leave for a few seconds.
  2. Tip off the excess crystal violet into the sink using forceps to hold the slide. Try not to get stains on your fingers, some of them are carcinogenic.
  3. Flood the slide with Gram’s iodine and leave for a few seconds more.
  4. The next step is the only one in which timing is critical. Wash out the crystal violet/iodine complex with acetone or acetone/alcohol by running the solvent down the slide. Stop when the purple colour stops running out of the smear. This will only take 1-2 seconds. IMMEDIATELY wash with running tap water.
  5. Place the slide back on the staining rack and flood the smears with counterstain, neutral red or safranin with or without added carbol fuchsin. Leave the counterstain on the slides for about 30 seconds.
  6. Wash well in running tap water and blot with pieces of blotting paper. Leave to dry thoroughly. All water must be removed from the smear before oil is added for examination in the microscope. If any water remains, blurry patches will be seen obscuring parts of the smear. Discard blotting paper in the contaminated waste bins (with the gloves) as there may still be viable organisms on the slide, particularly if the smear is made too thick.
  7. Examine the smear using the oil immersion lens on the microscope. You will not see the characteristics of the organisms properly under any other lens.
  8. First place the dry smear on the stage and focus the microscope using the 10X objective.
  9. Swing the objectives out of the way, and add a drop of oil to the smear.
  10. Bring the oil immersion objective down into the oil. If the smear was properly in focus first, it should be very close to being in focus for this lens and should only require a slight adjustment of the fine focus to see the organisms. If you cannot see any organisms, scan around, while at the same time “jiggling” the fine focus.

RESULTS

Gram positive organisms Dark blue/purple
Gram negative organisms Red/pink

NOTES

  • Older cultures of gram positive organisms may appear gram negative as their cell walls age and lose the ability to retain the crystal violet dye complex. Dead cells also appear gram negative.
  • Some gram positives do not stain uniformly and appear granular. This happens particularly with some species of Bacillus, and they may be mistaken for chains of cocci.
  • Bacterial spores do not take up any gram stains and appear as clear patches within vegetative cells or as clear glistening ovoids if free.
  • Some organisms appear to have a variable gram reaction i.e. some cells are purple, others pink. This may be due to aging or dead cells but some species are genuinely gram variable.
  • Protein and animal cells generally only take up the counterstain and appear pink.
  • Yeast cells generally take up the crystal violet and appear gram positive but as they are not bacteria, this term is not applicable. Fungal hyphae also stain with crystal violet.

     Gram stain procedure video

ZIEHL-NEELSEN STAIN

Acid fastness is a feature of some bacteria with specialized lipids in their cell walls. Examples include the Mycobacteria, which because of a cell wall containing mycolic acid, do not stain well with the gram stain. In this technique, stain (carbol fuchsin) is driven into the cell wall by mild heating and the presence of phenol in the stain reagent. The cells are washed in cold tap water which traps the stain in the cell. A mixture of acid (HCl) and ethanol is applied which removes the stain from most bacteria but not from those which are “acid-fast” such as the Mycobacteria. The remaining material is then counterstained. The technique can be modified by the use of less concentrated solutions of acid/alcohol as a decolourising agent in order to stain weakly acid-fast organisms such as Mycobacterium leprae in tissue sections and is also modified by using only acid as a decolouriser for organisms such as Nocardia spp. which are acid but not alcohol fast. . Variations also exist in which a highly concentrated solution of carbol fuchsin is used, removing the necessity for heating (Kinyoun stain).

PROCEDURE

  1. Prepare a heat fixed smear of a culture or clinical material.
  2. Place on the stain rack and flood the entire slide with carbol fuchsin.
  3. Gently heat the underside of the slide with a spirit burner flame (see notes) until steam begins to rise from the slide. DO NOT ALLOW TO BOIL. If the slide begins to get too hot, remove the flame for a few seconds. Keep the slide steaming for 7-10 minutes but do not allow to dry out. Add more stain if necessary.
  4. Wash well with cold tap water.
  5. Flood the slide with 3% HCl in 100% ethanol for 10 minutes, changing the solution at least 3 times.
    Wash in tap water.
  6. Counterstain with 5% w/v aqueous malachite green or 0.5% w/v methylene blue for 30 seconds.
    Wash in tap water.
  7. Blot dry and clean underside of slide with tissue. Examine with the oil immersion lens.

RESULTS

Acid Fast bacilli red/magenta
Non acid-fast organisms green or blue depending on counterstain

NOTES

  • There are significant safety issues involved in this procedure, however, if performed correctly and precautions observed, the technique is quite safe. Heating the phenol-containing dye solution is considered hazardous. Appropriate safety precautions should be observed i.e safety glasses should be worn and adequate ventilation should be available.
    An alternative heating method is to prepare a long rod by wrapping a large wad of cotton wool around one end of the shaft of a glass rod. This is then dipped briefly in ethanol (not dripping wet), ignited and used to heat the slide. The advantage is that the operator is further removed from the staining area and can avoid phenol fumes and there is no danger from the spirit burner which can ignite if not used correctly.
  • Methylene blue counterstain is generally recommended to avoid issues with operators who may be red/green colour blind.
  • Not all organisms may be displaying acid fast reactions and generally the slide must be scanned extensively to detect those organisms which are acid fast. This applies particularly with clinical material.
  • For weakly acid fast organisms, use 0.5% HCl in 100% ethanol as the decolourising agent. For Nocardia, use 1% HCl or H2SO4. 

Acid fast stain video

FLUORESCENT ZIEHL-NEELSEN STAIN

This technique was developed to facilitate scanning of clinical specimens for acid fast bacteria as the organisms are seen as fluorescent yellow/orange organisms against a dark background. This means there is more contrast and the organisms are more readily detected than in a conventional Ziehl-Neelsen stain. The technique also avoids heating of a phenol-containing dye solution. One disadvantage is that a fluorescence microscope is needed.

PROCEDURE

  1. Prepare a heat fixed smear of a culture or clinical material.
  2. Stain with auramine-phenol for 10 minutes.
  3. Wash with tap water
  4. Flood the slide with 3% HCl in 100% ethanol for 5 minutes.
  5. Wash with tap water.
  6. Apply 0.1% w/v KMnO4 for 30 seconds to quench background. Counterstaining is not necessary.
  7. Wash and blot dry.

RESULTS

Acid fast bacteria bright yellow fluorescence
Non acid fast bacteria not visible

SPORE STAIN

Production of endospores is a feature of some bacteria, particularly the large gram positive bacilli such as Bacillus spp. and Clostridium spp. Position and size of the spores is often used as an aid in identification of these species. As such, the spores must be visualized in the cells but spore cell walls are thick and generally do not stain in a gram stain. Heat is used to drive the stain into the spore walls in the following technique.

PROCEDURE

  1. Place a small beaker of water on a tripod over a Bunsen burner and bring the water to the boil.
  2. Prepare a thin heat fixed smear of culture.
  3. Place on the beaker, smear uppermost.
  4. When water begins to condense on the underside of the slide, flood the slide with 5% w/v aqueous malachite green.
  5. Leave for 3-6 minutes with the water constantly boiling.
  6. Remove the slide with forceps and wash with cold tap water over the sink.
  7. Counterstain at room temperature with 1% w/v aqueous safranin or 0.05% w/v aqueous basic fuchsin for 30 seconds.
  8. Wash and blot dry.

RESULTS

Spores green
Vegetative cells red/magenta

NOTES

  • Broth cultures give best results, but timing is crucial. If the culture is too young, no spores will have been produced, but if it is too old, all the spores will have been released from the vegetative cells and their size and position cannot be determined. Incubation between 24-48 hours is suitable for Bacillus subtilis.
  • Some species do not readily produce spores e.g. Clostridium perfringens rarely produces visible spores, especially in clinical material. Hence this staining technique may not be definitive for spore production.
  • In a gram stain, the area of the cell containing the spore can be seen as a clear zone.

Spore stain procedure video

ALBERT’S STAIN FOR METACHROMATIC GRANULES

Metachromatic granules are particles of polyphosphate found as a storage substance in certain bacteria, notably in Corynebacterium diphtheriae and some Mycobacteria, and are also known as volutin granules. They stain with methylene blue in Albert’s technique due to the multiple negative charges on the phosphates (metachromasia). For C. diphtheriae, optimal production of the granules occurs after growth on Loeffler’s slopes.

PROCEDURE

  1. Prepare a heat fixed smear.
  2. Apply Albert’s stain and allow to react for 3-5 minutes.
  3. Apply Gram’s iodine and allow to react for one minute.
  4. Wash in tap water and blot dry.
     

RESULTS

Metachromatic granules blue/black
Remainder of cell green

WET INDIA INK PREPARATION FOR CAPSULES

Many bacteria and some yeast cells produce a large capsule of carbohydrate surrounding the cell. For some organisms, production of a capsule can be indicative of a pathogenic strain (e.g. Streptococcus pneumoniae) or the capsule can be used as a means of identification (e.g. Cryptococcus neoformans). India ink preparations are basically negative stains where the ink particles are seen in the background while the capsules are seen as a clear halo around the organisms.

PROCEDURE

  1. Place a drop of a liquid culture of the organism on a slide or emulsify a colony in saline on the slide.
  2. Mix the container of India ink thoroughly.
  3. Dip a loop into the India ink and withdraw, taking care NOT to produce a film of liquid in the lumen of the loop.
  4. Mix the ink on the loop into the liquid on the slide.
  5. Place a coverslip on the slide and press down to make a thin film.
  6. Examine under low power to focus and then under high power (40x) to examine the capsules.

RESULTS

Encapsulated organisms display a black background with clear halos around black organisms.
Non-encapsulated organisms cannot be seen.

NOTES

  • Do not pick up too much India ink as nothing will be seen.
  • Avoid dry or clumping India ink solutions.

India Ink preparation video

 

Last modified: Monday, 5 February 2018, 10:18 AM