In this PPT I have explained processing of sputum sample step by step, first I have discussed about different methods of sputum sample collection and then also discussed about different criteria of sputum sample rejection after gram stain . in this PPT we will learn better about six different criteria of sputum sample rejection . In case of tuberculosis what is the criteria of sputum sample collection and processing also have been covered in this PPT. I hope it will be helpful for you thank you so much

1. Arpita CHANDRA

2. Types of sputum sample collection
1. Expectorated
2. Induced
3. Bronchoscopy
4. Endotracheal or Tracheostomy suction specimens
5. Transtracheal Aspirates
6. Other invasive procedure

3. Expectoration
 Good sputum samples depend on through health care
worker education and patient understanding all
phases of the collection process.
 Before Expectoration- Food should not have been
ingested for 1 to 2 hours
 Mouth should be rinsed with saline or water just
before expectoration

4. Sterile container
leak proof screw cap container
Specimen should be deep coughed
Transported to laboratory immediately

5. INDUCED
Who are unable to produce sputum may be assisted by
respiratory therapy
By using postural drainage and thoracic percussion to
stimulate production of acceptable sputum

6. Aerosol induced specimens
Useful for Isolation of Mycobacterium or fungal disease
- high diagnostic yeild
- In case of Pneumocystis jiroveci pneumonia as well

7. Induced specimen collection
By allowing the patient to breath
Aerosolized droplets of a solution containing 15% NaCl
and 10% glycerin for 10 min
Untill a strong cough reflex is initiated
These specimens are usually adequate for culture

8. Gastric aspirates
 Isolation of Acid fast bacilli
 Early morning sample
 A nasogastric tube is inserted into the stomach and
contents are withdrawn
 Resistance of Mycobacterium to
acidity sample must be delivered
immediately so that acidity can be
neutralized then processed

9. Endotracheal suction specimens
 Especially patient on ventilator
 Patient with trachiostomy rapidly colonized with gram
negative bacilli (P. aeruginosa and A. Baumannii)

10. COLLECTION
It should be done with a sterile technique using 22 inch
suction catheter , through the endotracheal tube
- First aspirates discarded
- Second aspirates should be collected after tracheal
instillation of 5 ml saline in a mucous collection tube

12. Bronchoscopy
By Bronchoalveolar lavage (BAL)
(most suitable for detecting Pneumocystis cysts and
fungal elements)

13. Procedure
In this procedure high volume of saline( 100-300ml)
infused into a lung segment though the bronchoscope to
obtain cells of the pulmonary interstitium and alveolar
spaces send a portion of it to the laboratory.

14. Trans-tracheal Aspirates (TTA)
 Invasive procedure
 Percutaneous transtracheal aspirates are obtained by
inserting a small plastic catheter into the trachea via
needle previously inserted through the skin and
cricothyroid membrane

15.  It cannot be used in uncooprative patients
 Rarely used anymore anaerobes- Actinomyces and
those associated with aspiration pneumonia
Other invasive Procedures
 When pleural empyema is present
 Thoracentesis may be used to
obtain infected fluid for direct
examination and culture

16. Specimen Processing
Lower respiratory tract specimen
Examined by
Direct wet mount- For parasite
Microscopy with 10%KOH – For fungal elements
Gram stain for Bacterial identification
AFB stain for M. tuberculosis

17. Ova of Paragonimus
westermannii
Budding yeast cells - Candida
Larvae of strongloides stercoralis

18. Sputum examination ( Macroscopic )
Examine for Colour, Consistency and odor
Appearance / odor Bacterial infection
Mucopurulent sputum Bacterial pneumonia
Scanty, watery sputum Atypical pneumonia
Rusty sputum Pneumococcal pneumonia
Current jelly or dark red K. Pneumonia
Foul smelling sputum Anaerobic infection
Greenish color Pseudomonas

19. Infection Indication
 Leukocytosis- Bacterial pneumonia
 Leukopenia- Viral infection
 Eosinophilia- Parasitic infection

20. Gram stain of sputum specimen
Acceptable specimens
Less than 10 squamous epithelial cells / low power
field (100x) WBCs may not be relevant, because many
patients are severely neutropenic .

21. On the other hand
Presence of 25 or more polymorphonuclear leukocytes /
field with few epithelial cells – Excellent specimen

22. Rejection criteria of ETAs
-Greater than 10 sqamous epthelial cells / field
-In Legionella pneumonia, sputum may be scant and
watery with no host cells such specimen may be positive
by direct fluoresent antibody stain and culture and
should not be subjected to screening procedure.

23. Sputum sample rejection criteria
1. Bartlett score
2. Murray and washington grading system
3. Gecklet et al
4. Van Scoy
5. Barry
6. Heineman and Radano2

24. Bartlett score
 Based on microscopic examination
1. Number of Neutrophils / field
2. Number of sqamous epithelial cells / field
(+) Score = acceptable sample
(-) Score = Non acceptable sample

25. 2. Murray and washington grading system
 Based on no. of epithelial cell / 10x field
 < 10 Epithelial cell / 10x field = acceptable
3. Geckler criteria
< 25 Epithelial cell / 10x field = acceptable
4. Van Scoy
Avg no of WBC/LPF
>25 WBC/LPF = Acceptable sample

26. 5. According to Barry
Assign (+) and(–)values
+3 (if >150 WBC) Any positive score= Acceptable
+2(If 76-150 WBC)
+1 (if 16-25 WBC)
Per 100x field
-3( if >25 EPC )
-2 (if 16-25 EPC)
-1 (if 5-15 EPC) / 100x field

27. 6. Heinman and Radano
 Avg ratio of WBC to EPC (> 1 EPC / every 10 WBC) in
the average low power field

28. In case of tuberculosis
 Sputum sample collection
- Early morning and spot ( according to NTEP)
 Concentration by
- Petroff method and modified petroff method by
adding NALC-NaOH
- Acid fast stain
- Fluorescent microscopy
- LJ culture

29. Modified Petroff method according to Belly & Scott
REAGENT PREPARATION
A (NALC-NaOH preparation)
(4%) NaOH (50mL)
Trisodium citrate (2.94%)(50ml)
NALC powder (0.5g)
Mix, sterile and store the NaOH and the citrate in sterile,
screw capped flasks for later use. This solution should be
used within hours after the NALC is added.

30. B - Phosphate Buffer preparation
Solution-A
 Sodium monohydrogen phosphate (anhydrous) 9.47 g
 Distilled water 1000ml
Solution- B
 Potassium dihydrphosphate 9.07g
 Distilled water 1000ml
Add 50 ml of solution B to 50 ml of solution A and
adjust pH to 6.8

31. Procedure
Transfer a maximum of 10 ml of sputum
In plastic 50ml conical centrifuge tube
Add equal volume of freshly prepared digestant (SOL-A) to the
tube
Votex the specimen for approximately 15 sec,
Mix it till the specimens are being digested
An extra pinch of NALC crystals may be necessary to liquefy
mucoid sputa
After 15 min. of digestion

32. Add enough phosphate buffer to reach within 1 cm of the top
Invert the tube to mix the solution, stop digestion process
Centrifuge all the tubes at 3600 x g for 15 min
Using aerosol free sealed centrifuge caps
Carefully pour off the supernatant into a splash proof
container

33. The lip of the tube may be wiped with an amphyl or phenol
soaked gauze to absorb drips
Resuspend the sediment in 1 to 2 mL phosphate buffer, pH
6.8 buffer (with bovine serum albumin)
Inoculate the sediment to culture media and prepare slide

34. Grading of AFB Smear
Number of AFBs seen Result and grading Number of field to
be examined
None in 100 fields Negative 100
1-9 in 100 fields Scanty 200
10-99 in 100 fields 1+ 100
1-10 per fields 2+ 20
Greater than 10
bacilli/field
3+ 20

35. Routine culture
 Mac Conkey agar - GNB
 Chocolate agar - Heamophilus and Neisseria species
 Do not inoculate on Enrichment broth and Anaerobic
culture
 Only percutaneous respiration and bronchial brush
are suitable for anaerobic culture
 Fro streptococcus pneumonia- with 5 % CO2 growing
best on blood agar also can grow on chocolate agar
 Manitol salt agar - S.aureus
 Lowenstein jensen media – M. tuberculosis

36. Reference
1. Bailey & Scott
2. Lester K. Wong et al. Comparison of six different
criteria for judging the acceptability of sputum
specimen.Journal of clinical microbiology
2019:16(4):0095