ACID
FAST STAINING TECHNIQUES PRINCIPLE: Most of the bacteria can be stained with aqueous solutions of para rosaniline dyes but certain organisms especially which belongs to family mycobacteriaceae resist entry of these weaker dyes and hence these weak solutions are unsuitable for staining them.However they can be stained with strong solutions which contain phenols which help in penetration of the in to the cell. Heating further enhances entry of the dye in to the cell. These organisms once stained resist decolorization with acids such as H2SO4,HCL,HNO3 etc. and even with alcohol.Those organisms which resist decolorize with acid retain primary stain and are called acid fast organisms and those which are decolorized by acid will takee up counter stain and are called non acid fast organisms. Reason for acid fastness is due to the presence of long chain fatty acid ,mycolic acid in the cell wall of these organisms
METHODS OR TECHNIQUES: There are various methods to demonstrate acid fast organisms. Important among them are as follows 1.Ehrlich
Method 2.Ziehl-Neelsen’s method 3.Gabbet,s method 4.Kinyoun,s method 5.cooper,s modification 6.Flouroscent staining method Ehrlich discovered acid fast staining technique in 1882 and was modified by Ziehl-Neelsen which is most widely used for demonstration of acid fast organisms EXAMPLES FOR ACID FAST ORGANISMS: 1.Mycobacterium tuberculosis 2.M.leprae 3.M.smegmatis 4.Atypical mycobacteria 5.Nocordia 6.Actinomycetes 7.Bacterial spores 8.Cryptosporidium parvum 9.Isospora bellii ZIEHL-NEELSEN’S TECHNIQUE: REQUIREMENTS: 1.Clinical specimen such as sputum ,CSF,urine etc. 2.microscopic
slide 3.bunsen burner 4.staining set -strong carbol fuchsin -20% H2SO4 -Loffler’s methtylene blue 5.microscope PROCEDURE: 1.prepare a
smear on a microscopic slide from the clinical sample 2.keep the slide on the staining rack 3.flood the slide with strong carbol fuchsin for 5-7 minutes with intermittent heating until steam rises . avoid boiling or drying of the stain. 4.wash with water 5.decolorise with 20% H2SO4 for 2 minutes 6.wash with water 7.add counter stain loffler’s methylene blue for 30 seconds 8.wash with water 9.blot dry the slide and observe under oil immersion objective Observation: red colored,rod shaped ,slightly curved
bacilli are seen against bluish background of pus cells and epithelial cells Inference: the given smear shows one of the acid fast organism GRADING OF THE SMEAR: OBSERVATION GRADE 0 bacilli per 300 fields no acid fast organism found 1-3 bacilli per 300 fields doubtful, repeat the smear 1-9 bacilli per 100 fields 1+ 1-9 bacilli per 10 fields 2+ 1-9 bacilli per field 3+ > 10 bacilli per field 4+ SIGNIFICANCE OF GRADING: 1.TO know the severity of the clinical condition 2.to assess the prognosis of the clinical condition NOTES: 1.Concentration of H2SO4 used is different depending on the organism suspected.20% for M.tuberculosis, 5% for M.leprae, 1% for nocordia,actinomycetes and .25-.5% for bacterial spores 2.M.tuberculosis is both acid and alcohol fast while M.smegmatis is only acid fast. This helps in differentiating them in urine samples where M.smegmatis occurs as commensal in urinary tract. 3.malachite green or picric acid can also be used as counterstains 4.Ziehl-Neelsen technique is also called hot method as heating is employed while Gabbet’s and Kinyoun’s methods are called cold methods as heating is not employed 5.In cold methods concentration of the dye and phenol is increased and staining is done for prolonged period 6.Atleast 10000 bacilli per ml of the sputum should be present to get positive results in microscopuy. In such situations concentration of the bacilli should be done by concentration techniques such as petroff’s method before smear preparation 7.csf and urine samples should always be concentrated before smear preparation as direct examination is very less sensitive
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