“QUALITY CONTROL OF AFB AND GRAM STAIN”

By

ARCHANA NAGAR &

GIRISH TANK

CONTENTS:-1. Quality assurance2. Quality control3. Quality control of AFB staining4. Quality control of Gram staining

Quality assurance:

It is a system which assure that the overall activities in the laboratory are being performed as designed by particular standards.

Quality control:Quality control deals with the system which accepts or rejects any activities or parameter which affects the quality of product and thus prevent quality deficiency.

QUALITY CONTROL OF ACID FAST STAINING

A control slide should be included with each run of stain this will verify the correct performance of the procedure as well as the staining intensity of the acid fast organisms.SLIDE PREPARATION:- QC slides may be prepared as follows-1. Prepare seprate suspension approximately

equal to a no . 1 Mc farland turbidity standard of

E.coli ATCC 25922(negative control) and mycobacterium tuberculosis ATCC H37RU

(positive control) in approximately 1.0 ml of physiological saline or sterile water in a biological safety cabinet.

McFarland StandardsMcFarland turbidity standards are used to standardize the approximate number of bacteria in a liquid suspension by visually comparing the turbidity of a test suspen-sion with the turbidity of a McFarland standard.

2. LABLING:-lable control slides with name(eg. AFB control,like positive and negative control).Record date of preparation and preparers name appropriately.

3. Using a insulin syring or loop place a drop of the negaive control suspension near the end of the glass slide.

4. Repeat step 3 with the suspension of the positive control ,but place the drop half way between positive negative control and the slide lable.

5. Allow smears to air dry in the biological safety cabinet.6. Heat fix the smears.7. Store control slides in a labeled slide box.

Staining Procedure:-8.Flood slide with Carbol Fuchsin9.Hold a flame beneath the slide until steam appears but do not allow it to boil10.Allow hot slide to sit for 3 to 5 minutes, rinse with tap water11.Flood slide with acid fast decolorizer12.Allow to sit 1 minute, rinse with tap water13.Flood slide with Methylene Blue14.Allow to sit 1 minute, rinse with tap water15.Blot dry16.View under oil immersion lens

Acid Fast Staining

EVALUATION:-

1. Control slide should be reviewed before the patient smears are read to confirm that the mycobacteria stain acid fast.

a. Carbolfuchsini. mycobacterium tuberculosis

(ATCC H37RU ): red or magenta against a blue background

ii. E.coli (ATCC 25922): blue.

2. RECORD THE RESULTS OF THE CONTROL SLIDE IF IT IS ACCEPTABLE , GO ON TO THE PATIENT SMEARS. IF THE CONTROL SLIDE IS UNACCEPTABLE , REVIEW PROCEDURES AND REAGENT PREPARATION. UNACCEPTABLE CONTROL SLIDES INCLUDE THE FOLLOWING :

b.carbolfuchsin i. positive control is not stained red ii.negative control remains red after

decolorization iii.background is not properly decolorized.

C. When the problem is resolved,re-stain a control slide as well as all patients slides from the problem run.d. Record the QC slide results on the appropriate log sheet.Fill out the cosecutive action if there are any of the above mentioned problem.

Gram stained control slides must be verified before examining and reporting patient smears. Manufacturer or user prepared QC slides can be used. If preparing your own QC slides, 18- to 24-hour cultures of known gram-positive and gram-negative organisms are used. The manufacturer’s product insert will state the organism name and american type culture collection (ATCC) strain number that is recommended.

QUALITY CONTROL OF GRAM STAINING

CELL WALL DIFFERENCEs

1. Prepare seprate suspension approximately equal to a no . 1 Mc farland turbidity standard of

SLIDE PREPARATION:- QC slides may be prepared as follows-

Positive control: Staphylococcus aureus (ATCC 25923)

Negative control: Escherichia coli(ATCC 25922)

in approximately 1.0 ml of physiological saline or sterile water in a biological safety cabinet.

2. LABLING:-lable control slides with name(eg. GRAM control,like positive and negative control).Record date of preparation and preparers name appropriately.3. Using a insulin syring or loop place a

drop of the negaive control suspension near the end of the glass slide.

4. Repeat step 3 with the suspension of the positive control ,but place the drop half way between positive negative control and the slide lable.

5. Allow smears to air dry in the biological safety cabinet.

6.Heat fix the smears.7. store control slides in a labeled slide box.

Staining Procedure:-8.On a rack, flood with filtered crystal

violet 10 sec9. Wash briefly in water to remove

excess crystal violet10. Flood with Gram’s iodine 10 sec11. Wash briefly in water, do not let

the section dry out. 12.Decolourise with acetone until the

moving dye front has passed the lower edge of the section

13. Wash immediately in tap water14. Counterstain with safranin 15

seconds

QUALITY CONTROL:-A. Check appearance of reagents daily If crystal violet has precipitate or crystal sediment ,

refilter before use .B. Daily and when a new lot is used , prepare a smear of

E.coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923)E.coli (GNB - Pink color)Staphylococcus aureus (GPC - deep violet color)

Gram Staining – Gram +’ve

Dr.T.V.Rao MD 21

Gram Staining – Gram -’ve

Dr.T.V.Rao MD 22

Good Quality Poor Quality

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