MICROBIOLOGY LAB 1 and 2 – Staining USTMED ’07 Sec C – AsM; Photos provided by JV.N. & A.M.

Stain – coloring of organisms with a dye that emphasizes certain structures Smear – a thin film of material containing the microorganisms spread over the surface of the slide Fixation – a procedure done before staining a smear to: 1. attach the microorganisms to the slide 2. kill the microorganisms 3. preserve the various parts of microbes in their natural state with only minimal distortion 3 Methods of Fixation 1. Air drying 2. Passing over flame of Bunsen burner 3. cover slide with methyl alcohol for 1 minute if not fixed, stain may wash the microbes


Decolorizer – a chemical to remove the primary stain - Ex. Gram Stain 95% C2H2OH (acetone alcohol) Acid fast stain Acid alcohol Secondary stain – a chemical which imparts a contrasting color to the primary stain - Ex. Gram stain Safranin Acid fast Methylene blue, malachite green Spore safranin stain


Stains – salts composed of a positive and a negative ion

1. 2.

basic dyes (chromatophore) – positive ion acidic dyes – negative ion

I. Gram Stain Purpose Reagents Primary Crystal violet stain Mordant Grams iodine Decolorizer Secondary stain 95% C2H2OH safranin

Bacteria – slightly negative charge; pH 7 Add (+) ion [basic dye] to charge bacteria = attract Ex. Crystal violet, methylene blue, safranin Add (-) ion [acidic dye] to charge bacteria = repel Ex. Eosin, nigrosin, acid fuchsin Negative stain – prepare colorless bacteria against a colored background Importance: 1. valuable in the observation of overall shapes, sizes and capsules 2. distortion of cell size and shape are minimized a. heat fixing is not necessary b. cells do not pick up the stain Types of stain 1. Simple stain Purpose – cellular shape and structure is made visible

Gram + Violet or purple Dark violet or purple Violet or purple Violet or purple

Gram Violet or purple Dark violet or purple Colorless red

Gram stain of Micrococcus species. Microscopically, micrococci are larger than staphylococci and appear in tetrads rather than grapelike clusters.

Urethral discharge with PMN and intracellular gram negative diplococci suggestive of Neisseria gonorrhoeae.

2. -

Ex. Methylene blue, carbol fuchsin, crystal violet, safranin Differential stain Distinguishes two groups of organisms or different parts of a bacterial cell Ex. a. Gram stain b. Acid fast stain i. Ziehl Neelsen stain ii. Modified Kinyoun stain c. Spore stain

i. ii.

Principle: 1. When applied to both grampositive and gram negative cells, crystal violet and then iodine readily enter the cells. 2. Inside the cells, the crystal violet and iodine combine to fome CV-1 which is larger than the crystal violet molecule that entered the cells.

Schaefer Fulton method Wirtz Conklin method

3. 4.

In Gram positive cells, crystal violet-iodine complex is trapped and therefore, retains the color. In Gram negative cells, the alcohol disrupts the lipopolysaccharides and the CV-I complex is washed out through the thin peptidoglycan layer. 5. Gram negative cells are colorless until counterstained with safranin after which they are pink. Clinical Significance - Provides valuable information for the treatment of disease Gram Penicillin + Cephalosporin Gram More resistant because the antibiotics do not penetrate the lipopolysaccharides II. Acid Fast Stain Purpose Reagents Primary Carbol stain fuchsin Mordant Steam or phenol Decolorizer Acid alcohol Secondary Methylene stain blue or malachite green Gram + Red Red Red Red Gram Red Red Colorless Blue or green

Reagents in Differential stain 1. Primary Stain – imparts color to all cells Ex. Gram stain Crystal violet Acid fast stain Carbol fuchsin Spore stain Malachite green 2. Mordant a. b. c. – a chemical added to primary stain to: Intensify Increase affinity To coat a structure to make it thicker

- Types of mordant o Physical mordant – steaming o Ex. Ziehl Neelsen stain Schaefer Fulton method Wirtz Conklin method o Chemical mordant o Ex. Gram stain Flagellar stain Modified kinyoun stain

Grams iodine Tannic acid phenol

Modified Kinyoun acid-fast stain. Nocardia species appear acid-fast when stained with a modified Kinyoun stain using 2% H2SO4 as the decolorizing agent. This feature helps to distinguish this mircroorganism from other actinomycetes. Prinicple 1. Acid fast organisms retain the red color because the carbol fuchsin is more soluble in the cell wall lipids than in the acid alcohol. 2. Non-acid fast organisms – cell walls lack the lipid components; carbol fuchsin is rapidly removed during decolorization Mycolic acid – a cell wall component responsible for the acid fastness of the organism Acid fast bacilli – organisms which are hard to stain but once stained, they are hard to decolorize Ex. Genus Mycobacteria, Genus Nocardia Clinical significance the finding of acid fast bacilli in a sputum is a presumptive evidence that the organisms is Mycobacterium tuberculosis. III. Special stains or selective stains – stains used to color and isolate specific parts of microorganisms such as endospores, flagella, capsules and metachromatic granules A. Negative staining for capsules 1. India ink technique - mix the bacteria in a solution containing a fine colloidal suspension of colored particles. - then stain with a simple stain, safranin. - Result – Halos surrounding each stained bacterial cell. Capsule stain. The cell is the purple rod in the center of the clear area. The purple color is from the basic stain, crystal violet. The clear area is the capsule, and the background is colored by the negative, acidic stain (India ink). D. Gram Stain Purpose Primary stain Mordant Decolorizer Secondary stain Reagent crystal violet Grams iodine 95% C2H2OH Safranin red

Spore stain of Bacillus cereus. The arrows are pointed at green spores in a pink vegetative cell.

Spores Violet or purple Dark violet Colorless colorless

Vegetative cell Violet or purple Dark violet or purple Violet or purple Violet or purple

Gram stain of Bacillus cereus. The arrow is pointed at a spore, which is clear inside the gram-positive vegetative cell.

Metachromatic granules Ex. Neisser stain, Loefflers methylene blue stain Loefflers methylene blue stain. Corynebacterium diptheriae Demonstrate metachromatic granules when stained with Loeffler methylene blue stain or Neisser stain. The stain is best performed on colonies grown on a Loeffler agar slant. Metachromatic deposits are reddish purple in Loeffler methylene blue stain.

- fin Demo slides….. Vibrio spp. (curved bacilli)


Flagella stain – a tedious and delicate staining procedure using a mordant and the stain carbol fuchsin to build up the diameters of the flagella until they become visible. Vibrio cholerae. Monotrichous flagella polar flagellum – one

Gram + stain

Proteus. Peritrichous flagella – flagella over the entire bacterial cell

Streptococcus lactis (cocci in chains)

C. -

Endospore (spore) staining – both selective and differential Ex. Schaefer Fulton method, Wirtz Conklin method Reagent Malachite green Steam Tap water safranin Spores Green Green Green green Vegetative cell Green Green Colorless red Sarcinae lutea (cocci in tetrads)

Purpose Primary stain Mordant Rinse Secondary stain

Congo red staining

4. Once cool, the slide is transferred to a support over a sink and flooded with a stain called Gentian Violet (a dye consisting of a methyl derivative of pararosaniline). The stain is left on the slide for about 1 minute. This stains all the bacteria on the slide a dark purple colour. Note, this stain will not penetrate the waxy cell walls of some bacteria eg mycobacteria. 5. The Gentian gently washed off the slide with running water Violet is

India Ink

6. The bacterial smear is then treated with Gram’s solution which consists of 1 part iodine, 2 parts potassium iodide, and 300 parts water. This iodine solution reacts with the Gentian Violet turning it a very dark shade of blue. It also causes it to be retained by certain types of bacteria in a way which is not really understood. 7. After about 30 seconds the slide is gently rinsed with ethyl alcohol which causes the dye-iodine complex to be washed out of some bacteria but not others. This is called decolourisation. If we now look at the smear down a microscope, the bacteria which had retained the Gentian Violet-iodine complex will appear blue-black. These are called Grampositive. However wi would not be able to see those which had lost the dye-iodine complex which are called Gramnegative. The final step in the gram stain method is, therefore, to stain the Gram-negative cells so they can be seen.

Malachite green staining

S. aureus

8. This is achieved by treating the smear with a compound which stains the Gramnegative cells a colour which contrasts markedly with the blue-black colour of the gram-posiitve cells. The stain common used for this is either eosin or fuchsin, both of which are red. These are called counterstains. Bacteria in the smear which are Gram-positive are unaffected by the counterstain. 9. The counterstain is left on the smear for about 30-60 seconds and then gently rinsed away with running water.

Bacillus subtillis (gram positive bacilli in chains)

Streptococcus lactis (cocci in chains)

10. After the counterstain has been rinsed off, the slide is placed between some absorbent paper and the excess water gently blotted off. Care must be taken not to rub the slide with the blotting paper because this would remove the adhering bacteria. The Gram staining method 1. A small sample of a bacterial culture is removed from a culture. In this example it is being taken from a broth culture of the pure microbe but it could be removed from a culture on solid medium or from material containing bacteria eg faeces or soil. 2. The bacterial suspension is smeared onto a clean glass slide. If the bacteria have been removed from a culture on solid media or it is from a soil or faeces sample it will have to be mixed with a drop of bacteria-free saline solution. 3. The bacterial smear is then dried slowly at first and the, when dry, heated for a few seconds to the point when the glass slide is too hot to handle. This fixes ie kills the bacteria making the slide safe to handle. Care must be taken not to overheat which will char the cells. 11. The slide is gently warmed to dry off any residual moisture and then a drop of oil immersion oil is placed on the stained bacterial smear. This helps transmit light through the specimen directly to the high-powered microscope lens. 12. The slide is the placed on a microscope stage and the oil-immersion lens lowered into the immersion oil. High-powered lenses are required because bacteria are very small (hindi nga???!!!) Clinical specimen : Urethral discharge Staining used: Gram staining Results: Gram neg cocci in pairs Intracellularly located Most possible microorg : Neisseria gonorrhea Pseudomonas aeruginosa

Stain used: Gram Staining Results: Gram Negative bacilli in singly/random (red slender rods in singly or random) Streptococcus pyogenes Stain used: Gram Staining Results: Gram Positive Cocci in chains (violet round/spherical cocci in chains) Escherichia coli Stain used: Gram stain Gram reaction: Gram negative (red) Morphology: Coccobacilli arragned singly or random Note: E. coli is a Gram Negative Bacilli but it appears as a short plump bacilli so it is called coccobacilli (see arrow) Staphylococcus aureus. Gram stain of culture showing characteristic irregular clusters of gram-positive cocci. There are no spores or capsules.

1. the flame for 3 times

Fix smear by passing slide over

2. Cover the smear with enough Kinyoun’s Carbol Fuchsin stain (initial stain) for 2-3 minutes. 3. Rinse with tap water


flood the smear with 3% acid alcohol (decolorizer) until the smear appears colorless or until only a suggestion of pink remains (30-45 secs). rinse with tap water

6. cover the smear with methylene blue (counter stain) for 20-30 seconds) 7. rinse with tap water

8. air dry or blot with filter paper then examine the slide under oil immersion lens. Report as follows: More than 10 AFB per filed 1-10 AFB per field 10-100 AFB per slide 1-9 AFB per slide report report report report +++ ++ + the exact number of AFB

Mycobacterium tuberculosis Stain used: Kinyoun’s Acid fast stain Clinical specimen used: Sputum Results: Positive for acid fast bacilli. acid fast stain of direct smear to show acid fast bacilli staining deep red (arrow A) and non-acid fast bacilli cells staining blue with the counter stain methylene blue (arrow B)

Bacillus subtilis Stain used: Gram stain Results: Gram positive Bacilli in chain (violet rods in chain) Aerobic Sporeformer Bacilli Note: Spores elliptical and centrally located SPORES – unstained VEGETATIVE PORTION – violet Kinyoun’s acid fast stain. Mycobacteria are readily stained with carbol fuchsin, which binds the cycolic acid in their lipid-rich cell walls. This stain cannot be removed (decolorized) with acid alcohol and, therefore, the microorganisms are referred to as acid-fast. There are two common acid-fast stains, Ziehl-Neelsen (ZN), and Kinyoun’s. The difference is that the ZN stain requires heat during the staining process because the phenol concentration used is less than in the Kinyoun’s method. The decolorizer, acid alcohol, and the counterstain, methylene blue, are the same for both methods. When stained, acid-fast bacilli stain red and the background is blue.

Jayveeh Navarro Amelia Mendoza Auds Martinez audrey_cl@yahoo.com

Kinyoun’s Acid Fast Staining (Cold Method)

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