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

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EXERCISE 12: ACID FAST STAIN ACID FAST STAIN

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. Early detection of infection 2. 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 Number of AFB Observed per Oil Immersion Field (OIF) 0 0

+ n 1 9 /100 VF 1 + 10 99 / 100 VF 2 + 1 10 / OIF in 50 VF 3 + > 10 / OIF in 20 VF

* VF = Visual Field

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PROCEDURE

1. Heat fix the sputum smear provided.

2. Place a folded piece of filter paper strip on the smear.

3. Saturate the paper with Ziehls carbolfuchsin.

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4. 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.

5. 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.

6. Put methylene blue (counterstain) and let it stay for 2 minutes.

7. Rinse with tap water, blot dry.

8. 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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DEMO SLIDES

(Photo taken on August 10, 2012 vinC)

Acid-Fast Cell

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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

2. Name two (2) genera that are identified by the use of acid-fast staining procedure. @ Mycobacterium @ Nocardia

3. What is the composition of the outer waxy coating surrounding bacteria that are acid-fast? @ Fatty acids, most specifically mycolic acids

4. 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

5. 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).

3. Place swabs in a sterile tube and immediately transport the specimen to the laboratory.

4. Label the specimen properly.

(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.

3. 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)

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.)

3. Examine plates for signs of growth and do a Gram stain on whatever growth is on the medium.

4. Continue to identify your isolate by performing any or all of the following tests:

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Organism to be Identified Tests

Staphylococci and Streptococci Catalase

Staphylococcus aureus Coagulase

Group A Streptoccoci Bacitracin

To differentiate Streptococcus pneumonia & viridans streptococci

Optochin Growth Inhibition, Bile Solubility

Gram-positive, non-sporeforming bacilli Virulence; animal inoculation, Elek

Neisseria Oxidase, Carbohydrate fermentation

Non-fermentative Gram-negative bacilli Oxidase, Citrate Utilization, Urease, Motility, Carbohydrate fermentation, Gelatin Liquefaction

Enterobacteriaceae Carbohydrate fermentation, Citrate Utilization, Methyl Red Vogues Proskauer Urease, Lysine decarboxylase,

Phenylalanine deaminase, Motility, Gelatin Liquefaction

Mycobacteria Niacin, Nitrate Reduction, Catalase

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REVIEW QUESTIONS

1. What organisms are considered normal throat flora? What organisms are considered pathogenic in the throat?

Normal Flora Pathogenic spp. Actinomyces, Bacteriodes, Micrococcus, Enterococcus, Fusobacterium, Lactobacillus, Neisseria, Staphylococcus, Streptococcus, Treponema, Proteus

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.

3. 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

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

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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. Instruct patient to cough deeply to expectorate a purulent specimen.

3. 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.

4. Label specimens properly and transport promptly to the laboratory.

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. Stain one smear with Gram stain, the other with acid fast stain.

3. 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

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c. Loefflers medium for Corynebacterium d. Chocolate agar for meningococci and Haemophilus e. Bordet-Gengou medium for Bordetella f. Lowenstein-Jensen medium for Mycobacteria g. Thioglycollate medium

2. Incubate the above plates in the following manner: a. BAP, MacConkey, Thioglycollate, Bordet-Gengou, Lowenstein-Jensen media are incubated at 35o C

for 18-24 hours. b. Chocolate agar is incubated in a candle jar or CO2 incubator overnight.

3. Examine the plates and tubes for signs of growth and do a Gram stain on whatever growth is on the medium.

4. Continue to identify your isolate by following the appropriate procedures.

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OBSERVATIONS

Culture of Sputum Specimen

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

1

2

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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

DEMO SLIDES

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 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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DEMO SLIDES

Paragonimus westermani, Ova

Paragonimus westermani, Adult

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REVIEW QUESTIONS

1. What is the specimen of choice for the recovery of P. westermani? @ Stool @ Sputum

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2. 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

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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