MICROBIOLOGY LAB 1 and 2 – StainingUSTMED ’07 Sec C – AsM; Photos provided by JV.N. & A.M.USTNotesGroup2009Updated 2006 - A. Abad

Stain – coloring of organisms with a dye that emphasizes certain structuresSmear – a thin film of material containing the microorganisms spread over the surface of the slideFixation – a procedure done before staining a smear to:

1. attach the microorganisms to the slide2. kill the microorganisms3. preserve the various parts of microbes in

their natural state with only minimal distortion

3 Methods of Fixation1. Air drying2. Passing over flame of Bunsen burner3. cover slide with methyl alcohol for 1 minute

- if not fixed, stain may wash the microbes

Stains – salts composed of a positive and a negative ion

1. basic dyes (chromatophore) – positive ion2. acidic dyes – negative ion

Bacteria – slightly negative charge; pH 7

Add (+) ion [basic dye] to charge bacteria = attractEx. Crystal violet, methylene blue, safranin

Add (-) ion [acidic dye] to charge bacteria = repelEx. Eosin, nigrosin, acid fuchsin

Negative stain – prepare colorless bacteria against a colored background

Importance:1. valuable in the observation of overall

shapes, sizes and capsules2. distortion of cell size and shape are

minimizeda. heat fixing is not necessaryb. cells do not pick up the stain

Types of stain1. Simple stain- Purpose – cellular shape and structure is

made visible- Ex. Methylene blue, carbol fuchsin, crystal

violet, safranin2. Differential stain- Distinguishes two groups of organisms or

different parts of a bacterial cell- Ex.

a. Gram stainb. Acid fast stain

i. Ziehl Neelsen stainii. Modified Kinyoun stain

c. Spore staini. Schaefer Fulton methodii. Wirtz Conklin method

Reagents in Differential stain1. Primary Stain – imparts color to all cells

- Ex.Gram stain Crystal violetAcid fast stain

Carbol fuchsin

Spore stain Malachite green

2. Mordant – a chemical added to primary stain to:

a. Intensifyb. Increase affinity c. To coat a structure to make it thicker

- Types of mordanto Physical mordant – steaming

o Ex. Ziehl Neelsen stainSchaefer Fulton methodWirtz Conklin

method

o Chemical mordant o Ex.

Gram stain Grams iodine

Flagellar stain Tannic acid

Modified kinyoun stain

phenol

3. Decolorizer – a chemical to remove the primary stain- Ex.

Gram Stain 95% C2H2OH (acetone alcohol)

Acid fast stain

Acid alcohol

4. Secondary stain – a chemical which imparts a contrasting color to the primary stain- Ex.

Gram stain

Safranin

Acid fast Methylene blue, malachite green

Spore stain

safranin

I. Gram StainPurpose Reagents Gram + Gram -Primary stain

Crystal violet

Violet or purple

Violet or purple

Chemical Mordant

Grams iodine

Dark violet or purple

Dark violet or purple

Decolorizer

Acetone Alcohol

Violet or purple

Colorless

Secondary stain

Safranin Violet or purple

Red

End Result Violet 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.

Principle:1. When applied to both gram-positive 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.

3. In Gram positive cells, crystal violet-iodine complex is trapped and therefore, retains the color.

4. 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 +

PenicillinCephalosporin

Gram -

More resistant because the antibiotics do not penetrate the lipopolysaccharides

II. Acid Fast StainPurpose Reagents Gram + Gram -Primary stain

Carbol fuchsin

Red Red

Mordant Steam or phenol

Red Red

Decolorizer

Acid alcohol

Red Colorless

Secondary stain

Methylene blue or malachite green

Red Blue or green

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.

Prinicple1. 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 capsules1. 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).

B. 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 – one polar flagellum

Proteus.

Peritrichous flagella – flagella over the entire bacterial cell

C. Endospore (spore) staining – both selective and differential

- Ex. Schaefer Fulton method, Wirtz Conklin method

Purpose Reagent Spores

Vegetative cell

Primary stain

Malachite green

Green Green

Mordant Steam Green GreenRinse Tap water Green ColorlessSecondary stain

safranin green red

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

Gram StainPurpose Reagent Spores Vegetative

cellPrimary stain

crystal violet

Violet or purple

Violet or purple

Mordant Grams iodine

Dark violet Dark violet or purple

Decolorizer

95% C2H2OH

Colorless Violet or purple

Secondary stain

Safranin red

colorless Violet or purple

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

D. Metachromatic granules- Ex. Neisser stain, Loefflers methylene blue stain

Loefflers methylene blue stain.Corynebacterium diptheriaeDemonstrate 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)

Gram + stain

Streptococcus lactis (cocci in chains)

Sarcinae lutea (cocci in tetrads)

Congo red staining

India Ink

Malachite green staining

S. aureus

Bacillus subtillis (gram positive bacilli in chains)

Streptococcus lactis (cocci in chains)

The Gram Stain Introduction

Gram’s Stain is a widely used method of staining bacteria as an aid to their identification. It was originally devised by Hans Christian Joachim Gram, a Danish doctor.

Gram’s stain differentiates between two major cell wall types. Bacterial species with walls containing small amounts of peptidoglycan and, characteristically, lipopolysaccharide, are Gram-negative whereas bacteria with walls containing relatively large amounts of peptidoglycan and no lipopolysaccharide are Gram-positive.

It’s a mystery

Although it may seem strange, the reason why bacteria with these two major types of bacteria cell walls react differently with Gram’s stain appears to be unconnected with the wall structure itself. The exact mechanism of the staining reaction is not fully understood, however, this does not detract from its usefulness.

The Gram staining method1. 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 unto a clean glass slide. If the bacteria have been removed from a culture or a solid media or it is from a soil or feces sample It will have to be mixed with a drop of bacteria-free saline solution3. 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.

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 Violet is gently washed off the slide with running water

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 Gram-positive. However wi would not be able to see those which had lost the dye-iodine complex which are called Gram-negative. The final step in the gram stain method is, therefore, to stain the Gram-negative cells so they can be seen.

8. This is achieved by treating the smear with a compound which stains the Gram-negative 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.

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.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

Clinical specimen :Urethral discharge

Staining used:Gram staining

Results:Gram neg cocci in

pairsIntracellularly

locatedMost possible microorg :

Neisseria gonorrhea

Pseudomonas aeruginosaStain used: Gram StainingResults:

Gram Negative bacilli in singly/random (red slender rods in singly or random)

Streptococcus pyogenesStain used:

Gram StainingResults:

Gram Positive Cocci in chains (violet round/spherical cocci in chains)

Escherichia coliStain used: Gram stainGram reaction:

Gram negative (red)Morphology:

Coccobacilli arragned singly or randomNote:

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.

Bacillus subtilisStain used: Gram stainResults: Gram positive Bacilli in chain (violet rods in chain) Aerobic Sporeformer BacilliNote: Spores elliptical and centrally locatedSPORES – unstainedVEGETATIVE 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.

Kinyoun’s Acid Fast Staining (Cold Method)i. Fix smear by passing slide over the flame for 3 times

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

iii. Rinse with tap water

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

v. rinse with tap water

vi. cover the smear with methylene blue (counter stain) for 20-30 seconds)

vii. rinse with tap water

viii. air dry or blot with filter paper then examine the slide under oil immersion lens.

Manner of Reporting/Recording for AFB Smear Results (2001) IUATLD SystemNo AFB in at least 100 fields

0 or Negative

1-9 AFB in 100 Fields + n (report the actual AFB count)Suggest repeat collection

10-99 AFB per 100 field +11-10 AFB/field in at least 50 fields

+2

>10 AFB/field in at least 20 fields

+3

Mycobacterium tuberculosisStain used:

Kinyoun’s Acid fast stainClinical specimen used:

SputumResults:

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)

Jayveeh NavarroAmelia MendozaAuds Martinezaudrey_cl@yahoo.com

Gram stain of Clostridium paraputrificum (x1250). This microorganism displays terminal, swollen spores and the gram-variable staining typical of Clostridium species. Spores may not take up the stain, so they may appear as clear areas. (terminal spore)Sputum smear stained with Gram’s stain shows neutrophil amorphous debris, and filamentous, beaded, branched gram-positive bacilli (oil immersion)Gram stain of pus from an empyema cavity showing long and short chains of Gram-positive streptococci and large numbers of pus cells (stained red). X3,900

large boxcar shapped Gram (+) bacilli

Gram stain Nocardia spp. (x1250). Nocardia species are branching, beaded, filamentous gram-positive bacilli, approximately 1 um in diameter. They can also appear as coccoid or coccobacillary forms. Care should be taken when examining the slides because of the faint staining properties of these microorganisms and other actinomycetes.Flagella stain, Proteus sp.

Sputum stained with Gram’s stain shows many neutrophil amorphous debris, and coryneform gram-positive bacilli (oil immersion)

Demo Slides

Diplococcus pneumonia

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Gram (-) Bacilli Gram (+) Cocci

Loeffier methlene blue stainCorynebacterium diphtheriaemetachromatic granules

Sarcina luteaGram positive tetrad

Sprillum (spiral bacterium) Urethral smear report

Vibria comma (curved bacilli)

Lab 2 addition

India Ink Method(Staining of Capsule)Indirect or negative or relief staining1. To a small loopful of water on a clean glass slide, ad a minute amount of growth from the agar culture by using a sterile inoculating needle. (Broth culture maybe used directly.)2. Mix well, then add a small drop of INDIA INK and immediately cover with a thin coverslip allowing the fluid to spread as a thin film beneath the coverslip.3. Examine under the high power objective and then with the oil immersion lens. Regulate the light with the sunstage condenser of the Iris diaphragm.

Klebsiella pneumoniaeCapsule stain

Congo Red Method(Staining of Capsule)Indirect or negative or relief staining

1. Using a sterile inoculating needle fish out a small amount of colony of Klebsiella pneumoniae and mix it with a drop of CONGO RED stain and spread 2 cm. Diameter.2. Air dry. Do not heat.3. Cover the smear with 1% ACID ALCOHOL until the smear turns blue.4. AIR DRY and examine under Oil Immersion lens.

Malchite Green Method(Staining of Spores)

1. Prepare smear from bacillus subtilis2. Flood the slide with 5% AQUEOUS SOLUTION OF MALACHITE GREEN and heat to gentle steaming for 2 or 3 minutes. Avoid overheating and drying.3. Pour off excess stain and rinse thoroughly with tap water.4. Flood the smear with SAFRANIN for 30 seconds5. Rinse again with tap water, blot dry and examin under Oil immersion objective.

SPORE: GreenVegatative Portion: Red

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

Kinyoun’s acid fast stain (x1500). Mycobacteria are readily stained with carbol fuchsin, which binds the mycolic 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 strains, 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, as shown here.

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