Many mycobacterial culture samples are heavily contaminated with commensal flora. It is critical to remove these because they can outgrow the culture faster than the mycobacteria.
Principle of the Petroff’s Concentration Method
Contaminant destruction is dependent on exposing the sample to a chemical agent that will kill them without inhibiting Mycobacteria growth. The majority of decontamination procedures employ acids such as sulfuric, hydrochloric, or oxalic acid, or alkalis such as sodium hydroxide or tri-sodium phosphate. Sodium hydroxide (4%) is a useful decontaminating agent for sputum, biopsies, body fluids, and so on, and should be followed by washing with sterile Milli Q water.
Type of Sample used
Sputum, urine, biopsy, and other bodily fluids that may be contaminated with commensal flora.
Equipment and Reagent
- Laminar flow hood (Class III)
- Light microscope
- Test specimen
- Dehydrated media (Lowenstein Jensen (LJ)
- 4 % Sodium hydroxide-sterilize by autoclaving use-within a week.
- Sterile Milli Q water
- Other lab wares
Sample Management and Instructions
- Sputum: After rinsing the mouth with water, instruct the patients to collect deeply coughed, expectorated sputum in a sterile wide-mouthed red-top plastic container.
- Urine: Instruct the patients to collect the first voided (early morning) samples in the laboratory’s sterile container. Urine collection instructions state that the genitals and surrounding area should be cleaned with moist cotton and copious amounts of water to avoid contamination by normal flora.
- Label the specimen with the patient’s identification number and the nature of the specimen.
Petroff’s sputum/urine concentration method Procedure
- The processing of the specimen must take place in the biological safety cabinet level III.
- Inoculate the sputum/clinical specimen over the surface of the LJ medium with a sterile loop. Close the slopes tightly, incubate at 37°C, and label the slopes as needed. Make a smear from the specimen and stain it with Ziehl Neelsen to detect AFB.
- Centrifuge the entire urine sample and inoculate the deposit onto the LJ medium.
- Smear the deposit (for urine) before decontaminating it with sodium hydroxide and staining it with the Ziehl-Neelsen method.
- Add two volumes of sterile 4 percent sodium hydroxide to each volume of remaining sputum/urine/other specimens, taking care to avoid contact between the specimen bottle rim and the sodium hydroxide flask.
- Shake the centrifuge tubes by hand for one minute.
- Place in a rack on the shaking machine (wrist action shaker) and shake for 20 minutes.
- Remove the specimen from the shaker.
- Centrifuge for 15 minutes at 3000 rpm
- After removing the supernatant from the centrifuge, carefully pour it into the disinfectant jar.
- Fill the centrifuge tubes up to 3/4 full with sterile Milli Q water, shake by hand to mix the deposit, centrifuge at 4000 rpm for 15 minutes, and then pour off the supernatant as before. Repeat the washing process two more times with sterile Milli Q water.
- Finally, use a loop to inoculate sediment onto two previously labeled Lowenstein-Jensen slopes.
- Place the inoculated L J slants in the 37°C incubator.
- Using the deposit, make a thin smear. Allow it to dry inside the hood and stain with the Ziehl-Neelsen method
- Examine the slopes on a weekly basis for signs of growth. Incubate for another 6-8 weeks before generating negative results.
Procedure for Ziehl Neelson Stain
- A thin smear of the specimen should be prepared. Dry in the air, then gently heat for 2 minutes over a Bunsen burner flame to fix the smear.
- Flood the heat-fixed smear with carbol fuchsin slide, heat from below the slide until fumes appear, and leave for 5-7 minutes. Do not allow the stain to boil.
- Wash off the stain with distilled water
- Cover the smear with 20 % sulfuric acid for 3 minutes
- Wash well with distilled water.
- Decolorization is repeated until the smear appears pale pink.
- Cover the slide with methylene blue for 2 minutes.
- Wash with distilled water, dry and examine the slide under oil immersion microscope.
Ziehl-Neelsen stain Results Interpretation
If any definite bacilli (pink/red rods) are observed, the smear should be reported as acid fast bacilli (AFB) positive, with an indication of the number of bacteria present as follows:
|More than 10 AFB/field||Report as 3 + (plenty)|
|1–10 AFB/ field||Report as 2+ (many)|
|10–100 AFB/100 fields||Report + (moderate)|
|1–9 AFB/100 fields||(few)|
Limitation of Ziehl-Neelsen stain
Ziehl-Neelsen stain cannot determine the exact species of Mycobacteria. It can only differentiate whether the bacteria is acid fast or not. Further tests must be performed to identify the species of Mycobacteria.
- Follow the usual precautions as for handling any biological fluid
- Wear disposable gloves and face mask while doing the procedure
- Temperature: 25 to 30°C.
Laboratory strategy in the diagnosis of infective syndromes. Mackie & McCartney. Practical Medical Microbiology. Edited by Collee, Dugid, Fraser, Marmion. 1989;14. 600-50.