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EXERCISE 12: ACID FAST STAIN ACID FAST STAIN

Subject: Microbiology Laboratory Topic: Exercises 12 - 15 Transcriptionist: vinC Pages: 14

Developed in 1882 by Paul Ehrlich First demonstrated on Mycobacterium bacilli Used to detect organisms that contain relatively large amount of lipid material (mycolic acids) in their cell wall Only Mycobacteria and Nocardia species produce cell wall-bound mycolic acids. ZIEHL-NEELSEN STAIN A modified Ehrlich technique Usage of heat to allow the primary stain (carbolfuchsin) to enter the lipid-containing cell wall 5% phenol used as chemical intensifier of dyes entry into the cell KINYOUN ACID-FAST METHOD More commonly used today Higher concentration of phenol in the primary stain solution makes intracellular penetration of carbolfuchsin better Heating is not needed Referred to as the cold method Acid-fast organisms resist decolorization hence, appearing red or pink Non-acid fast organisms absorb methylene blue stain (counterstain) KINYOUN ACID-FAST METHOD More commonly used today Higher concentration of phenol in the primary stain solution makes intracellular penetration of carbolfuchsin better USES OF ACID-FAST STAIN 1. 2. Early detection of infection Monitoring infection

System of reporting of smear which is recommended by Smithwick and David and has been modified by the Lung Center of the Philippines Interpretation 0 +n 1+ 2+ 3+ * VF = Visual Field Number of AFB Observed per Oil Immersion Field (OIF) 0 1 9 /100 VF 10 99 / 100 VF 1 10 / OIF in 50 VF > 10 / OIF in 20 VF

_____________________________________________________________________________________________ PROCEDURE 1. 2. 3. Heat fix the sputum smear provided. Place a folded piece of filter paper strip on the smear. Saturate the paper with Ziehls carbolfuchsin.

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Heat the slide directly over the flame. The stain should steam but never allowed to boil nor dry. Add just enough stain periodically during heating to prevent drying of the slide. The procedure is carried on for 5 minutes. Rinse slide with tap water for 30 seconds and decolorize with acid alcohol for about 15 seconds or until the solution runs clear. Rinse again with tap water. Put methylene blue (counterstain) and let it stay for 2 minutes. Rinse with tap water, blot dry. In examining under oil immersion field, select one straight imaginary line dividing the smear into two equal halves as seen in the diagram below.

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2 cm.

3 cm.
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Acid-Fast Cell

(Photo taken on August 10, 2012 vinC)

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_____________________________________________________________________________________________ REVIEW QUESTIONS: 1. What color do acid-fast bacteria stain? Non-acid fast? Acid-fast bacteria stain red Non-acid-fast bacteria stain blue Name two (2) genera that are identified by the use of acid-fast staining procedure. Mycobacterium Nocardia What is the composition of the outer waxy coating surrounding bacteria that are acid-fast? Fatty acids, most specifically mycolic acids How does heating the bacterial smear during a ZN stain promote entry of carbolfuchsin into the acid-fast cell wall? Heating melts the mycolic acid, thus allowing the stain to penetrate the cell wall What is the clinical value of acid fast stain? Identification of bacteria in the genus Mycobacterium, in which some are pathogens Useful in identifying acid-fast bacilli Rapid, preliminary diagnosis of tuberculosis and leprosy Performed on patient samples to track the progress of antibiotic therapy Performed on patient samples to determine the degree of contagiousness

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_____________________________________________________________________________________________ ADDITIONAL NOTES: Mycobacterium and Nocardia are those that test positive for acid-fast stain which may or may not be pathogens. The acid-fast stain is typically done on specimens suspected to contain Mycobacterium tuberculosis. Mycobacterium tuberculosis causes tuberculosis A positive acid-fast smear is used as a presumptive evidence of tuberculosis Growth on culture and identification procedures is done for the confirmation of tuberculosis. Mycobacterium species grow slowly (18-24 hr. doubling time), hence cultures takes 3 to 8 weeks for initial growth and disease confirmation.

EXERCISE 13: BACTERIOLOGIC WORK-UP OF THROAT SWAB SPECIMEN Throat and nasopharyngeral cultures are important in the diagnosis of infections o Ex. Streptococcal sore throat, scarlet fever, diphtheria, and whooping cough Useful in determining the focal point of infection in diseases o Such as rheumatic fever and acute glomerulonephritis Essential for detection of beta-hemolytic streptococci, hospital staphylococcus species, Corynebacterium diphtheria, and other potential pathogens.

NASOPHARYNGEAL CULTURES are recommended for pneumococcus, meningococcus, and Haemophilus influenza isolation NASOPHARYNGEAL SWABS are essential for recovery of meningococci from carries and for Bordetella pertussis from suspected cases of whooping cough.

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____________________________________________________________________________________________ PROCEDURE A. COLLECTION OF SPECIMEN 1. Collect throat swab prior to the start of antibiotic regimen under good lighting using a sterile swab. For this exercise, collect throat swab from one of your groupmates. 2. Depress the tongue with a tongue depressor, and rub the two swabs firmly over the back of the throat, both tonsils, crypts and any area of inflammation, exudation or ulceration. (Avoid touching the tongue, cheeks, teeth, or lips with swab). Place swabs in a sterile tube and immediately transport the specimen to the laboratory. Label the specimen properly.

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(If diphtheria is suspected, three swabs should be collected: one to be cultured onto Loefflers medium immediately, best done at the bedside or the out patient clinic; and the two others for culturing other organisms.)

B. MICROSCOPIC EXAMINATION 1. Using one of two swabs, make at least two smears per specimen, air dry and heat-fix them. 2. Stain one smear with Gram stain. If diphtheria is suspected, stain the other smear with metachromatic granules stain. Examine the smears under oil immersion lens and note the predominating types of organisms present like: a. Gram positive cocci in chains, pairs or clusters (Staphylococci, Streptococci) b. Gram negative cocci in pairs (meningococci) c. Gram positive rods (Corynebacteria) d. Gram negative rods (Bordetella, Haemophilus, Pasteurella, coliforms) e. Metachromatic granules (Corynebacterium)

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C. INOCULATION OF CULTURES 1. With the remaining swab, inoculate the following media: a. BAP for Staphlococci and Streptococci b. McConkey plate for Gram-negative rods c. Thioglycollate medium (Other media that could be used in clinical practice are: Loefflers medium for Corynebacterium; chocolate agar for meningococci and Haemophilus; Bordet-Gengou medium for Bordetella; and Lowenstein-Jensen medium for Mycobacterium) 2. Incubate the above plates at 35 0C for 18-24 hours. (If Chocolate agar is used, it must be incubated in a candle jar or CO2 incubator overnight.) Examine plates for signs of growth and do a Gram stain on whatever growth is on the medium. Continue to identify your isolate by performing any or all of the following tests:

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Organism to be Identified Staphylococci and Streptococci Staphylococcus aureus Group A Streptoccoci To differentiate Streptococcus pneumonia & viridans streptococci Gram-positive, non-sporeforming bacilli Neisseria Non-fermentative Gram-negative bacilli Enterobacteriaceae

Tests Catalase Coagulase Bacitracin Optochin Growth Inhibition, Bile Solubility Virulence; animal inoculation, Elek Oxidase, Carbohydrate fermentation Oxidase, Citrate Utilization, Urease, Motility, Carbohydrate fermentation, Gelatin Liquefaction Carbohydrate fermentation, Citrate Utilization, Methyl Red Vogues Proskauer Urease, Lysine decarboxylase, Phenylalanine deaminase, Motility, Gelatin Liquefaction Niacin, Nitrate Reduction, Catalase

Mycobacteria

_____________________________________________________________________________________________ REVIEW QUESTIONS 1. What organisms are considered normal throat flora? What organisms are considered pathogenic in the throat? Normal Flora Actinomyces, Bacteriodes, Micrococcus, Enterococcus, Fusobacterium, Lactobacillus, Neisseria, Staphylococcus, Streptococcus, Treponema, Proteus Pathogenic spp. N. meningitides and H. influenzae, B. pertussis C.diphtheriae K. pneumonia

Opportunistic Organisms: Staphylococcus aureus, S. pneumoniae, S. pyogenes, Mycobacterium gordonae, Actinomyces israelli, Streptococcus viridans, Fusobacterium necrophorum, Bacteriodes coagulans, Hemolytic streptococci 2. How reliable is Gram stain in the diagnosis of acute sore throat? It is not a reliable method in diagnosis, since many organisms that can be detected by Gram stain that may be not be the infective organism causing the inflammation. What incubation conditions are required for throat cultures? The covered plate is incubated at a temperature of 350 370 C for 24 to 48 hours to foster the growth of bacteria. If chocolate agar is used, it must be incubated in a candle jar or CO2 incubator overnight

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EXERCISE 14: BACTERIOLOGIC WORK-UP OF SPUTUM SPECIMEN Used for microbiologic diagnosis of lower respiratory tract infectinos Proper instructions and supervisions must be done to the patients in order to obtain a good quality of specimens provided by the patients

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Poor quality of specimens are usually brought about by: o Poor instructions given to the patients many times, saliva instead of sputum is submitted o During specimen collection, supervision is generally lacking Specimens are not delivered promptly to the laboratory Overgrowth of other bacteria and delay in the diagnosis happens due to late delivery of specimens

____________________________________________________________________________________________ PROCEDURE A. COLLECTION OF SPECIMEN 1. Patients should be instructed to rinse out their mouth with water or brush their teeth so as to remove contaminating food particles and secretions. 2. 3. Instruct patient to cough deeply to expectorate a purulent specimen. Collect sputum in a sterile, wide mouthed bottle or Petri dish, 1-3 mL is sufficient, except for culturing tubercle bacillus. For Mycobacteria, collect 3-single, early morning, 5-10 mL specimens on 3 successive days. In patients in whom expectorated sputum samples cannot be obtained, sputum induction using hypertonic sodium chloride solution will be helpful. Label specimens properly and transport promptly to the laboratory.

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B. MICROSCOPIC EXAMINATION 1. Using one of two swabs, make at least two smears per specimen, air dry and heat fix them. Select purulent or bloody flects, if present. 2. 3. Stain one smear with Gram stain, the other with acid fast stain. Examine under oil immersion lens. The first step in the examinations of a gram stained sputum is to determine the adequacy of the sputum submitted. Specimens with both >10 epithelial cells and <25 leukocytes per field may be reasonable rejected except when the patient is severely leukopenic. The presence of any of the following is a presumptive evidence of bacterial pneumonia: a. b. c. d. e. any encapsulated, lancet-shaped cocci occurring singly, in pairs or in chains along with pus cells suggests pneumococcal infection. great numbers of gram-positive cocci in cluster suggest staphylococcal pneumonia numerous short, fat, encapsulated Gram-negative rods suggest Klebsiella infection non-encapsulated gram negative rods suggests Pseudomonas the acid-fast stained smear is an important diagnostic aid since it is a presumptive evidence of tuberculosis. Presence of red, slender, slightly curved long or short rods is indicative of infection with Mycobacteria. NOTE: RESULTS OF STAINED SMEARS ARE ONLY PRESUMPTIVE AND THEREFORE SHOULD BE CONFIRMED BY CULTURE.

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C. INOCULATION OF CULTURES 1. With the remaining swab, inoculate the following media: a. b. BAP for Staphylococci and Streptococci MacConkey plate for Gram-negative rods

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Loefflers medium for Corynebacterium Chocolate agar for meningococci and Haemophilus Bordet-Gengou medium for Bordetella Lowenstein-Jensen medium for Mycobacteria Thioglycollate medium

Incubate the above plates in the following manner: a. b. BAP, MacConkey, Thioglycollate, Bordet-Gengou, Lowenstein-Jensen media are incubated at 35o C for 18-24 hours. Chocolate agar is incubated in a candle jar or CO2 incubator overnight.

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Examine the plates and tubes for signs of growth and do a Gram stain on whatever growth is on the medium. Continue to identify your isolate by following the appropriate procedures.

_____________________________________________________________________________________________ OBSERVATIONS Culture of Sputum Specimen

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Note: There are two different cultures that can be observed on Blood Agar Plate after a 24 hour incubation.

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Note: No culture is observed on MacConkey Agar. Indicative of the absence of a gram negative bacteria from the sputum sample.

Gram Staining of Cultures

Gram staining of the mucous-like colony (1) from BAP

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Gram staining of the green colony (2) from BAP

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Example of gram stained sputum specimen of a patient with a pathogen _____________________________________________________________________________________________

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REVIEW QUESTIONS 1. When are you going to reject a sputum specimen for culture? Macroscopic o sputum of less than 2mL unless obviously purulent o if saliva is given rather than sputum Microscopic o if there are >10 epithelial cells per low power field o if there are <25 leukocytes per low power field In a gram-stained smear from a sputum sample, epithelials are enumerated with the oil-immersion objective. Give two respiratory infections whose diagnosis is aided by microscopic examination of smear and culture of properly collected sputum specimen. a. b. 4. Bacterial Pneumonia (Pneumococcus) Tuberculosis

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Based on the microscopic examination of sputum smears stained by the Gram method, the presence of what organism is indicated by the following morphologic features: a. b. c. d. Gram-positive diplococci Gram-positive cocci in clusters Large Gram-negative rods Small Gram-negative rods Streptococcus pneumoniae Staphylococcus aureus Klebsiella pneumoniae Haemophilus influenza

_____________________________________________________________________________________________ ADDITIONAL NOTES:

SPECIMEN COLLECTION Prior to collection, the mouth should rinsed out with water in order to remove loose cells The patient should be standing, if possible or sitting upright in bed He or she should take deep breath to full the lungs, and empty them in one breath, coughing as hard and as deeply as possible

GOOD QUALITY SPECIMENS Quantify number and types of inflammatory cells Note presence of bronchial inflammatory cells Concentrate on areas with WBCs when looking for organisms Determine if there is a predominant organism (>10 per oil immersion field) o Semiquantitate and report organism with description o If no predominant organism is present, report mixed gram positive and gram negative flora

OTHER ORGANISMS WITH DISTINCT GRAM STAIN FROM SPUTUM SAMPLE Gram postivie in chains Streptococcus pyogenes Thin gram negative rods Pseudomonas aeruginosa

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Pointed gram negative rods Fusobacterium Branching, beaded gram positive rods Nocardia Gram negative diplococcic Moraxella catarrhalis

MOST COMMON CAUSES OF BACTERIAL PNEUMONIA Streptococcus pneumoniae Staphylococcus aureus Haemophilus influenza Klebsiella pneumonia Streptococcus pyogenes Moraxella catarrhalis

SOME ORGANISM WHICH ARE NOT DETECTED WITH ROUTINE BACTERIAL CULTURE OF THE SPUTUM SAMPLE Mycoplasma pneumoniae and Chlamydia pneumonia Legionella species Mycobacterium tuberculosis

CHARACTERISTICS OF THE SPUTUM IN PATIENTS WITH BACTERIAL RESPIRATORY TRACT INFECTIONS thick consistency (viscous) appeared discolored yellowish, greenish, grayish or rarely rusty or bloody may have an unpleasant odor

EXERCISE 15: LUNG FLUKE: Paragonimus westermani Paragonimus westermani is one of the most important parasites affecting human lungs. Acquired thru ingestion of insufficiently cooked crabs or cray fish infected with metacercariae. Adult worm can cause acute and subacute infections of the lung tissue. It may also wander erratically to other organs such as brain and skin. Paragonimiasis usually mistaken with tuberculosis symptoms due to hemoptysis Diagnosis is confirmed by finding the medium sized operculated ova in sputum Treated by praziquantel

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Paragonimus westermani, Ova

Paragonimus westermani, Adult _____________________________________________________________________________________________ REVIEW QUESTIONS 1. What is the specimen of choice for the recovery of P. westermani? Stool Sputum

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Briefly discuss the laboratory tests that are useful in the diagnosis of paragonimiasis. Specific Laboratory Tests o Isolation of the ova Finding the characteristic egg from the sputum or feces if swallowed Eggs can also be found in the pleural fluid and skin lesions o Serology Complete Fixation Test (CFT) Enzyme Immunoassay (EIA) Non-specific Laboratory Test o Intradermal Test Screening test o Chest X-ray Can be opacity, ring shadow, calcified spots or cloudy infiltrations Common in the basal part of the lungs More on the left upper lobe than the right upper lobe

_____________________________________________________________________________________________ ADDITIONAL NOTES: Morphological feature of Paragonimus westermanii ova o 85 x 55 o Broader operculated anterior end o Thickened posterior end Some organisms which are NOT detected with a routine bacterial culture of the sputum

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REFERENCES: Past Transcriptions, Online Articles, Laboratory Manual In Microbiology and Parasitology, Markell and Voges Medical Parasitology 8th ed.

What they rarely say in med schools is that studying for the board exams starts from your first day in first year med. - @medstudentsPH

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