Gram's Stain Pack

Gram’s Stain Pack HS802 Instructions for use v3 12.01.2017

Product identity (name) Intended Use

HS802F, HS802NR, HS802S Gram’s Stain Pack IVD, for professional use only. (synonyms: None) CAS-No: N/A Type (s) of material Pack Size 4 x 250ml Air dried heat fixed bacterial smears from culture or tissue sections. The culture can come from a thick Product composition suspension of a liquid culture or a pure colony from a plate suspended in water.

1. HS230 - Crystal Violet 250ml Contains <5% ethanol (CAS-No 64-17-5 ) Suggested Staining Procedure 1, 2 2. HS305X - Gram’s Iodine Diluent 240ml 3. HS305Y - Gram’s Iodine Concentrate 10ml Principle: Contains Potassim iodide The method is named after its inventor, the Danish <20% (CAS-No 7681-11-0) scientist Hans Christian Gram (1853–1938). Gram staining differentiates bacteria by the chemi- 4. HS310 - Gram’s Decolouriser 250ml cal and physical properties of their cell walls by de- Contains: 75% acetone (CAS-No 67-64-1); tecting peptidoglycan, which is present in a thick 25% Iso-Propanol (CAS-No 67-63-0) layer in Gram-positive bacteria. In the Gram stain,

Gram-positive bacteria retain the crystal violet dye,

while the counterstain (Safranin, Carbol fuchsin or

Neutral red ) added after the crystal violet gives all

Gram-negative bacteria a red or pink colouring. 5. i) HS160 - Carbol Fuchsin (Counterstain) 250ml Contains: <0.1% methanol (CAS-No 67-56-1); <1% phenol (CAS No 108-95-2) Handling and treatment before staining: Prepare thin smears using a loopful of a pure culture on grease free slides. Air dry and heat fix. or Important Note for Gram’s Iodine HS305X+Y: ii) HS570 - Neutral Red (Counterstain) 250ml The 10ml Gram’s Iodine Concentrate HS305Y must or be added to the 240ml Diluent HS305X and mixed iii) HS720 - Safranin (Counterstain) 250ml well before use to make 250ml Gram’s Iodine HS305X+Y. For further information see SDS. Technique: 1. Flood the heat fixed smears with Crystal Violet Handling and storage Oxalate (HS230). Allow to stain for up to 1 mi nute. Store in a dry, well-ventilated place and use before 2. Wash with tap water. expiry date. 3. Stain with Gram’s Iodine (HS305X+Y) for 1 Stable when kept in tightly stoppered plastic bottles minute. away from heat and at room temperature. 4. Wash with tap water.

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TCS Biosciences Ltd, Botolph Claydon, Buckingham, MK18 2LR United Kingdom t: +44 (0)1296 714 222, f: +44 (0)1296 714 806, e: [email protected] www.tcsbiosciences.co.uk Gram’s Stain Pack HS802 Instructions for use v3 12.01.2017

Technique continued: Additional Information

5. Decolourise with Gram’s Decolouriser (HS310) The thickness of the smear used in the Gram stain until no further stain runs from the slide. will affect the result of the stain. The step that is 6. Wash thoroughly with tap water. most crucial in effecting the outcome of the 7. Counterstain with Safranin O (HS720); Neutral stain is the decolourising step. Over- Red (HS570); or Carbol Fuchsin (HS160) for decolourising will lead to an erroneous result where up to 1 minute Gram-positive cells may stain pink to red indicating 8. Wash, air dry and examine. a Gram-negative result, and under-decolourising will lead to an erroneous result where Gram- negative cells may appear blue to purple indicating a Gram-positive result. Expected Results 1, 2 It is important to recognise that not all species of As shown below, organisms that retain the violet- bacteria can be usefully stained by Gram's method. iodine complexes after washing in decolouriser Some species are Gram variable and some are stain purple and are termed Gram-positive. Those called Gram indeterminant. I that lose the violet iodine complexes are termed If more or less intense staining is required the times Gram negative. of immersion in the various solutions should be al- tered accordingly.

Users must determine the suitability of the kit for their particular use.

Bibliography

1. Bergey, David H.; John G. Holt; Noel R. Krieg; Peter H.A. Sneath (1994). Bergey's Manual of Determinative Bacteriology (9th ed.). Lippincott Williams & Wilkins. ISBN 0-683-00603-7.

2. Beveridge T.J (March 1990). "Mechanism of Gram variability in select bacteria". Journal of Bacteriology 172 (3): 1609–20.

European conformity according to the "IN-VITRO DIAGNOSTIC MEDICAL DEVICES DIRECTIVE 98/79/EC, ANNEX III". Manufactured by TCS Biosciences Ltd.

v2 created 27.05.2016, change to classification of component '3'. v3 created 12.01.2017, addition of StainHD Logo. Page 2 of 2 IFU HS802 v3 12.01.2017