Page 238 - The Veterinary Laboratory and Field Manual 3rd Edition

Microbiology 207

routine and special stains used for 3 Flood the slide with crystal violet solution
microbiology smears and leave for 1 min.
4 Wash the slide, drain and flood with Gram’s
A Gram stain is routinely performed on bacte- iodine, leave for 1 min.
ria from cultures and on smears submitted for 5 Wash the slide, drain and add acetone or alco-
microbiological examination. This is because hol. Hold the slide under running tap water
the Gram stain reaction is an integral part of the until no further colour is removed (approxi-
routine bacterial identification procedure. For mately 30 s). Do not over-decolourize).
effective staining, it is important to make sure 6 Counter-stain with dilute carbol fuchsin
that smears prepared from specimens and cul- (leave for 1 min).
tures are not too thick and that cells are evenly 7 Wash the slide again in water and leave to dry
distributed. A Bunsen flame can be used to kill in a slide rack.
the bacteria and fix them to the slide. 8 When dry the slide can be examined under
the microscope using the oil immersion lens
(1000×).
Gram stain
GraM StaIn rEaGEntS Gram-positive organisms stain blue/purple
1 Crystal violet (stock solution: crystal violet and Gram-negative organisms stain pink in
1 g, water 200 ml. Dissolve by mixing and colour. It is useful to have reference cultures
filter before use). of Streptococci (Gram +ve) and Escherichia coli
2 Gram’s iodine (iodine 1 g, potassium iodide (Gram –ve) to check that the staining reagents/
2 g, water 300 ml). technique is working well (see page XX).
3 Acetone or methylated spirits (industrial
methyl alcohol). ModIFIEd zIEHL–nEELSEn StaIn.
4 Dilute carbol fuchsin (ZN carbol fuchsin Modified Ziehl–Neelsen stain is used to detect
diluted 1/10 with distilled water). As an ‘acid-fast’ bacteria, that is, Mycobacteria in fae-
alternative to carbol fuchsin, safranin (0.5%) cal samples and in smears made from tissues.
or neutral red (0.1%) can be used. The stain may also be used to detect cryptospo-
ridia in faecal samples.
It is important to avoid constant re-use of stain-
ing jars for a series of slides because bacterial rEaGEntS
organisms may be transferred from one slide to 1 Dilute 1/10 carbol fuchsin (basic fuchsin 1 g,
another giving false results. Most commercial absolute alcohol 10 ml, 5% phenol in 100 ml
Gram stain kits come with four small labelled of water).
bottles of reagents – a dropper bottle or a lidded 2 Acetic acid (0.5%).
bottle with a pipette are preferred. 3 Methylene blue (1%).

ProcEdurE ProcEdurE
1 Prepare a smear (from tissue swabs, sedi- 1 Heat fix the smear preparation (prepared
ments or from a culture plate) by mixing with from tissue, faecal swabs or fixed bacterial
1–2 drops of sterile water using a ‘flamed’ colonies).
wire loop. 2 Flood the slide with carbol fuchsin and leave
2 Heat fix the smear preparation on to the for 10 min.
microscope slide. 3 Wash the slide in water and drain.

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