Clinical Bacteriology Lab - 03 Microscopic Examination of Stained Preparation

CLINICAL BACTERIOLOGY LABORATORY POSITIVE STAINING MICROSCOPIC EXAMINATION IN STAINED PREPARATION MATERIALS OUTLINE • Introduction o Staining Techniques • Simple Stain o Introduction o Positive Staining o Negative Staining • Differential Stains o Differential Staining o Gram Staining o Acid Fast Staining • Special Stains o Introduction o Spore Stain o Capsule Stain o Flagellar Stain o Metachromatic Granule Stain INTRODUCTION STAINING TECHNIQUES • • Staining o Procedure that applies colored chemicals called dyes to the specimen in order to facilitate identification. Stains o are salts composed of positive and negative ion, one of which is colored (chromophore- color bearing ion), which imparts a color to a cell or cell parts and fixed to them through a chemical reaction. • Cultured organisms, clean glass slides, sterile inoculating loop, alcohol lamp or bunsen burner, bibulous paper, Safranin, Ziehl's Crbol Fuchsin, Loeffler's Methylene Blue, Crystal Violet or Gentian Violet PROCEDURE 1. 2. Get 3 clean glass slides and label samples 1, 2 and 3. By means of a sterile loop (heat to red-hot in alcohol lamp and allow to air cool) transfer a loopful of the bacterial suspension to the center of the slides. Re-flame the loop to kill the excess inoculum. Cool. 3. If transfer is made from solid media, a sterile loop is touched lightly upon the culture which is then transferred to a drop of water on a glass slide. Touch lightly and mix until a faintly turbid suspension results. 4. Spread the drop over the center of the slide and allow the film to air dry. 5. Fix the dried smear by passing the slide rapidly just over the flame, film side up, two or three times. 6. Flood the smear with several drops of the dye and allow it to remain for the following time intervals: (a) Sample 1 – Carbol Fuchsin = 15-30 seconds (stains mycolic acid) (b) Sample 2 – Crystal Violet = 30-45 seconds (c) Sample 3- Methylene Blue= 3-5 minutes 7. Wash in tap water to remove the excess stain, using a gentle indirect stream then air dry or use bibulous paper to blot. 8. Repeat with the other bacterial suspension. 9. Label all stained slides. 10. Examine the stained smears under the oil immersion objective. o Cedar wood oil can be thick LESSON 1: SIMPLE STAIN INTRODUCTION • • The simple stain can be used as a quick and easy way to determine cell shape, size and arrangements of bacteria. True to its name, the simple stain is a very simple staining procedure involving a single solution of stain. Any basic dye such as methylene blue, safranin, or crystal violet can be used to color the bacterial cells. These stains will readily give up a hydroxide ion or accept a hydrogen ion, which leaves the stain positively charged. Since the surface of most bacterial cells and cytoplasm is negatively charged, these positively charged stains adhere readily to the cell surface. After staining, bacterial cell morphology (shape and arrangements) can be appreciated. RESULTS AND OBSERVATIONS • The bacterial cells usually stain uniformly and the color of the cell depends on the type of dye used. If methyene blue is used, some granules in the interior of the cells of some bacteria may appear more deeply stained than the rest of the cell, which is due to presence of different chemical substances. NEGATIVE STAINING • • • In Negative staining technique, an acidic stain such as Nigrosin, India ink, Eosin or Congo red is used in which the bacterial culture or the specimen is mixed well and then spread over the Microscopic glass slide to form a thin smear. These stains do not penetrate and stains the bacterial cell due to repulsion between the negative charge of the stain and the negatively charged bacterial cell wall. Instead, these stains either produce a deposit around the bacteria or produce a dark background so that the bacteria appear as the unstained cell with a clear area around them. Hence in Negative staining background is stained instead of bacterial cells. LESSON 2: DIFFERENTIAL STAINS DIFFERENTIAL STAINING • • MATERIALS • Cultured organisms, clean glass slides, sterile inoculating loop, alcohol lamp, nigrossin solution or india ink PROCEDURE 1. 2. 3. 4. 5. Differential Staining is a staining process that uses more than one chemical stain. Using multiple stains can better differentiate between different microorganisms or structures/cellular components of a single organism. Differential staining is used to detect abnormalities in the proportion of different white blood cells in the blood. The process or results are called a WBC differential. This test is useful because many diseases alter the proportion of certain white blood cells. By analyzing these differences in combination with a clinical exam and other lab tests, medical professionals can diagnose disease. One commonly recognizable use of differential staining is the Gram Stain. Gram staining uses two dyes: Crystal Violet and Fuchsin or Safranin (the counterstain) to differentiate between Gram positive bacteria (large peptidoglycan layer on the outer surface of the cell) and Gram negative bacteria. Acid fast Stain is also a type of differential stain. Transfer a bacterial suspension on a slide. Add a drop of Nigrossin solution or India Ink to the bacterial suspension on the slide. Mix thoroughly and spread into a thin film to facilitate rapid drying. Do not fix it with heat. Examine the preparation under the OIO. Make a colorful drawing of representative bacteria observed in the stained smear to show the relative size, shape, and arrangement of the cells. Note the characteristic differences in the appearance of bacteria when stained by the various dyes. GRAM STAINING RESULTS AND OBSERVATIONS • INTRODUCTION/PRINCIPLE In Negative staining Preparations, the Bacterial cells observed as the Clear transparent bodies or objects, may be of variable size and shape if you are using a mixture of bacteria, against a dark background. • • • • o Vegetative Cells & Spores of Bacilli are Clearly Visible in Negative Stain Preparation The two major groups of bacteria can be divided into gram-positive and gram-negative. The Gram stain technique is based on the differential structure of the cellular membranes and cell walls of the two groups. Gram-positive organisms contain a highly cross-linked layer of peptidoglycan that retains the primary dye, crystal violet (CV), following the application of the mordant, iodine (I). The iodine and crystal violet form a complex within the peptidoglycan. When decolorizer is applied to the cells, the CV-I complex remains within the cell, making it appear dark purple to blue. The gram-negative organisms do not contain a thick crosslinked layer of peptidoglycan. The peptidoglycan is loosely distributed between the inner cell and the outer cell membranes. Following the application of the crystal violet and iodine, the CV-I complexes are not trapped within the peptidoglycan. Application of the acid-alcohol decolorizer dehydrates the outer cellular membrane, leaving holes in the membrane and effectively washing or removing the CV-I complex from the cells. The cells appear colorless. To make the colorless cells visible, a secondary stain, safranin, is applied, leaving the gram-negative cells pink. MATERIALS • o Negative Stain Preparation Showing Cocci in Clusters • Bacterial cultures, Clean glass slides, alcohol lam or bunsen burner, bibulous paper, inoculating loop, Crystal Violet, Gram's Iodine, 95 % Alcohol, Safranin. LIMITATIONS • • • • • • PROCEDURE 1. Prepare and fix the specimen to the microscope slide before staining. 2. Cover the smear with crystal violet, the primary stain, for 20 seconds. 3. Gently rinse off the stain with water. 4. Cover the smear with Gram’s iodine, the mordant, for 1 minute. 5. Pour off the excess Gram’s iodine. 6. Run the acid-alcohol decolorizer over the smear until the solution appears clear. 7. Gently rinse with water. 8. Cover the smear with safranin, the secondary or counterstain, for 20 seconds. 9. Gently rinse the stain with water. 10. Blot dry with bibulous paper. • • • Over-decolorization may result in the identification of false gram-negative results, whereas under-decolorization may result in the identification of false gram-positive results. Smears that are too thick or viscous may retain too much primary stain, making the identification of proper Gram stain reactions difficult. Gram-negative organisms may not decolorize properly. Cultures older than 16 to 18 hours will contain living and dead cells. Cells that are dead will be deteriorating and will not retain the stain properly. The stain may form a precipitate with aging. Filtering through gauze will remove excess crystals. Gram stains from patients on antibiotics or antimicrobial therapy may have altered Gram stain reactivity due to the successful treatment. Occasionally, pneumococci identified in the lower respiratory tract on a direct smear will not grow in culture. Some strains are obligate anaerobes. Toxin-producing organisms such as Clostridia, staphylococci, and streptococci may destroy white blood cells within a purulent specimen. Faintly staining Gram-negative organisms, such as Campylobacter and Brucella, may be visualized using an alternative counterstain (e.g., basic fuchsin). ACID FAST STAINING INTRODUCTION • Acid-fast mycobacteria contain mycolic acid in their outer membrane, making the cells waxy and resistant to staining with aqueous based stains such as the Gram stain. The primary stain, carbolfuchsin is applied to the cells, and heat or phenol/tergitol are used to allow the stain to penetrate into the waxy surface of acid-fast microorganisms. The excess stain is removed with treatment by acid alcohol (ethanol and hydrochloric acid). A secondary stain, methylene blue or malachite green, is then applied to the cells. ZIEHL-NEELSEN METHOD (HOT METHOD) • RESULTS • • • Gram-positive bacteria appear blue or purple Gram-negative cells will appear pink to red Examples of Gram-positive & Gram-negative bacteria: o Gram-positive bacteria: Staphylococcus aureus, Streptococcus pyogenes, Corynebacterium diphteriae o Gram-negative bacteria: Neisseria meningitidis, Escherichia coli, Pseudomonas aeruginosa • Materials o Sputum Sample, Clean glass slides, Inoculating loop, Alcohol lamp or bunsen burner, Pasteur pipet, Bibulous paper and Ziehl Neelsen reagents o Ziehl Neelsen Reagents: ▪ Primary Stain- Carbol fuchsin ▪ Mordant- Heat ▪ Decolorizer- Acid Alcohol ▪ Secondary Stain- Methylene Blue Procedure 1. Prepare and fix the specimen smear prior to staining. 2. Place a small strip of blotting or filter paper over the top of the specimen, and place the slide over a boiling hot water bath on a mesh surface. 3. Cover the filter paper with the primary stain, carbolfuchsin. Leave the slide on the water bath for 3 to 5 minutes. Continue to apply stain if the filter paper begins to dry. 4. Remove the filter paper and rinse the slide with water until the solution runs clear. 5. • • Run acid-alcohol decolorizer over the slide for approximately 10 to 15 seconds. 6. Rinse the slide with water. 7. Cover the smear with the secondary or counterstain, methylene blue, for 1 minute. 8. Gently rinse the slide with water. 9. Blot the slide dry with bibulous paper. Results o Acid fast: Bright red to intensive purple, Red, straight or slightly curved rods, occurring singly or in small groups, may appear beaded o Non-acid fast: Blue color; In addition, background material should stain blue Limitations: o The filter paper must remain moist and in contact with the specimen during heating to allow for proper penetration of the primary stain. o Organisms cultivated on blood agar may experience nutrient deprivation, resulting in a lower lipid content in the outer membrane resulting in poor staining. • REPORTING RESULTS OF ACID FAST BACILLI SMEARS Number of AFB* Seen Fuchsin Stain (1000x Magnification) 0 1-2/300 fields Number of AFB Report Seen in Fluorochrome Stain (450x Magnification) 0 No AFB Seen 1-2/60 fields Doubtful; request another specimen 1-8/100 fields 2-18/50 fields 1+ 1-9/10 fields 4-36/10 fields 2+ 1-9/field 4-36/field 3+ >9/field >36/field 4+ *AFB, Acid Fast Bacilli LESSON 3: SPECIAL STAINS INTRODUCTION • KINYOUN METHOD (COLD METHOD) • • • Materials o Sputum Sample, Clean glass slides, Inoculating loop, Alcohol lamp or bunsen burner, Pasteur pipet, Bibulous paper and Kinyoun's reagents o Kinyoun's Reagents: ▪ Primary Stain- Carbol fuchsin ▪ Mordant- Phenol/Tergitol ▪ Decolorizer- Acid Alcohol/ 1%Sulfuric Acid ▪ Secondary Stain- Malachite Green Procedure 1. Prepare and fix the specimen smear prior to staining. 2. Cover the smear with Kinyoun carbol fuchsin for 3 to 5 minutes at room temperature. 3. Gently rinse the slide with water. 4. Run 1% sulfuric acid decolorizer over the slide for approximately 3 minutes. 5. Rinse the slide with water and decolorize again for 1 to 2 minutes until the solution runs clear. 6. Rinse the slide with water. 7. Cover the smear with the secondary or counterstain, methylene blue, for 1 minute. 8. Gently rinse the slide with water. 9. Blot the slide dry with bibulous paper. Results o Acid-fast organisms, Mycobacterium spp., will appear pink. Note: Identification of a single acid-fast bacillus in single sputum is considered diagnostic. o Nonacid-fast organisms will appear blue-green. In addition, background material should stain blue. o Limitations o May be less sensitive than the Ziehl-Neelsen method. o Smears that are too thick may not properlystain. Special stain techniques seek to identify various bacterial structures of importance. It is used to examine bacterial spores, capsules, flagella, and metachromatic granules. For instance, a special stain technique highlights the flagella of bacteria by coating the flagella with dyes or metals to increase their width. Flagella so stained can then be observed. SPORE STAIN INTRODUCTION • Bacterial spores (endospores) are thick-walled, highly refractile bodies that are produced by Bacillus and Clostridium. The shape of the endospores and also their location within the vegetative cell vary depending on the species. Endospores are usually produced by cells growing in rich media but are approaching the end of active growth. Various factors such as aging or heat treatment are needed to activate the dormant spore. It permits them to undergo germination and outgrowth when they are placed in a suitable medium. MATERIALS • • • Bacillus subtilis (24 hr culture) Clean glass slide Spore stain (carbol fuchsin, 5% acetic acid, Loeffler’s methylene blue) METHOD: WIRTZ-CONKLIN • Procedure 1. Make a thin bacterial smear, air dry and fix. 2. Stain with 5% aqueous malachite green, steam for 6 minutes by heating over the Bunsen flame until vapor rises, cool. 3. Counterstain with 0.5% aqueous safranin for 30 seconds. 4. Wash with water. 5. Air dry. 6. Examine under OIO. • Result o The vegetative cell is red o The endospore is blue-green CAPSULE STAIN INTRODUCTION • Capsule is a thick gelatinous or slimy layer excreted by some bacteria outside the cells which is produced by the plasma membrane. This substance is also known as slimy layer or glycocalyx. It consists of complex sugar or polysaccharides which may combine with lipid and protein. The capsule is usually detected by special stain that stains the bacteria and the surrounding area. The capsule can also be demonstrated using a negative stain in which the bacteria and the capsule remain unstained against a black or dark background. Capsule helps the microorganism to glide, slide, or move on the surface of solid material. This substance also aid bacteria to attach themselves to mucous membrane and tooth surface which also contribute to its virulence by inhibiting phagocytosis. RESULT • • EXAMPLES OF CAPSULE POSITIVE AND NEGATIVE • • Klebsiella pneumoniae (24 hr culture) Clean glass slide Negative stain(India Ink, Congo Red, Nigrosin, or Eosin) Crystal Violet 20% aqueous copper sulfate PROCEDURE 1. 2. 3. 4. 5. 6. 7. Place a small drop of a negative stain (India Ink, Congo Red, Nigrosin, or Eosin) on the slide. Congo Red is easier to see, but it does not work well with some strains. India Ink generally works, but it has tiny particles that display Brownian motion that must be differentiated from your bacteria. Nigrosin may need to be kept very thin or diluted. Using sterile technique, add a loopful of bacterial culture to slide, smearing it in the dye. Use the other slide to drag the ink-cell mixture into a thin film along the first slide and let stand for 5-7 minutes. Allow to air dry (do not heat fix). Flood the smear with crystal violet stain (this will stain the cells but not the capsules) for about 1 minutes. Drain the crystal violet by tilting the slide at a 45 degree angle and let stain run off until it air dries . Examine the smear microscopically (100X) for the presence of encapsulated cells as indicated by clear zones surrounding the cells. Positive o Bacillus anthracis, Klebsiella pneumoniae, Streptococcus pneumonia Neisseria meningitidis Clostridium spp, Rhizaobium spp, etc. Negative o Neisseria gonorrhoreae, etc. FLAGELLAR STAIN INTRODUCTION • MATERIALS • • • • • Capsule: Clear halos zone against dark background No Capsule: No Clear halos zone Flagella are whip-like protein appendages which make bacteria move. Not all bacteria have flagella, however, for those that have, the number and the arrangement of the flagella are a characteristic of the specie. The flagella of bacteria arise from a basal body in the cell membrane and project outwardly through the cell wall and capsule. MATERIALS • • • • • Proteus vulgaris (24 hr culture) Clean glass slide Inoculating loop Alcohol lamp Leifson’s Stain METHOD: LEIFSON STAIN • Procedure 1. Prepare the stain by adding the following chemicals: ▪ Tannic acid 0.85 gms ▪ Sodium chloride 0.5 gms ▪ Pararosaniline 0.35 gms ▪ 95% ethanol 35 mL ▪ distilled water 65 mL 2. From an agar culture or single colony. Emulsify a loopful of bacteria in 5 mL of distilled water. 3. Using a new thoroughly cleaned slide flame one side and then allow to cool. 4. Place a large loopful of bacteria. Allow it to dry at room temperature. 5. . Flood the bacterial smear with Leifson stain for 8-10 minutes. 6. Wash the smear with tap water, drain and air dry. 7. Counterstain, if desired, with Loeffler’s Methylene blue for 1-2 minutes. 8. Wash with tap water and air dry. 9. Flagella stains red while the organism is blue. MECHANISM • • • • • • • First of all in this procedure thickness of flagella is increase so it can be visible. The Leifson’s stain is made up of tannic acid ,basic fuschin stain prepared in alcohol base. When we treat Leifson’s stain with cell the tannic acid get attach to the flagella and alcohol get evaporated. After evaporation of alcohol the thickness of flagella is increased due to deposition of tannic acid. Where as Basic fuschin stain the Flagella. After Leifson’s stain treatment cells are treated with Methylene blue stain. This Methylene blue stains the cell. RESULT • After observation under microscope we can observe that flagella appear red in colour and bacterial cell appear blue in colour. METACHROMATIC GRANULE STAIN INTRODUCTION • Also known as volutin granules, are composed of polyphosphate which are stained reddish-purple with methylene blue. These granules serves as a reserve source of phosphate that can be used by bacterial as a reserve energy source in case there is decrease nutrient available for microbial survival. MATERIALS • • • • • Corynebacterium diphtheriae culture Glass slide Tap water Iodine Albert’s stain METHOD: ALBERT’S STAIN • Procedure: 1. Prepare the smear, air dry and fix. 2. Flood the smear with Albert’s stain for 3-5 minutes. 3. Wash in tap water and drain off excess water. 4. Flood with Albert’s iodine for 1 minute. 5. Wash, air dry and examine under OIO. RESULT • • Granules appear as blue-black and the cytoplasm appears green.