Gram’s Stain Pack HS800 Instructions for use v1 06.03.2015 Product identity (name) Intended Use HS800F, HS800NR, HS800S 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. HS232 - Crystal Violet Oxalate 250ml Contains: ~1% methanol (CAS-No 67-56-1); Suggested Staining Procedure 1, 2 <2% crystal violet (CAS-No 548-62-9 ) <20% ethanol (CAS-No 64-17-5 ) Principle: The method is named after its inventor, the Danish scientist Hans Christian Gram (1853–1938). Gram staining differentiates bacteria by the chemi- 2. HS305X - Gram’s Iodine Diluent 240ml cal and physical properties of their cell walls by de- 3. HS305Y - Gram’s Iodine Concentrate 10ml tecting peptidoglycan, which is present in a thick 4. HS310 - Gram’s Decolouriser 250ml layer in Gram-positive bacteria. Contains: 75% acetone (CAS-No 67-64-1); In the Gram stain, Gram-positive bacteria retain the 25% Iso-Propanol (CAS-No 67-63-0) crystal violet oxalate dye, while the counterstain (Safranin, Carbol fuchsin or Neutral red ) added after the crystal violet oxalate gives all Gram- negative bacteria a red or pink colouring. 5. i) HS160 - Carbol Fuchsin (Counterstain) 250ml Handling and treatment before staining: Contains: <0.1% methanol (CAS-No 67-56-1); Prepare thin smears using a loopful of a pure cul- <1% phenol (CAS No 108-95-2) ture on grease free slides. Air dry and heat fix. Important Note for Gram’s Iodine HS305X+Y: or The 10ml Gram’s Iodine Concentrate HS305Y must be added to the 240ml Diluent HS305X and mixed ii) HS570 - Neutral Red (Counterstain) 250ml well before use to make 250ml Gram’s Iodine or HS305X+Y. iii) HS720 - Safranin (Counterstain) 250ml Technique: For further information see SDS. 1. Flood the heat fixed smears with Crystal Violet Oxalate (HS232). Allow to stain for up to 1 minute. Handling and storage 2. Wash with tap water. Store in a dry, well-ventilated place and use before 3. Stain with Gram’s Iodine (HS305X+Y) for 1 expiry date. minute. Stable when kept in tightly stoppered plastic bottles 4. Wash with tap water. away from heat and at room temperature. 5. Decolourise with Gram’s Decolouriser (HS310) until no further stain runs from the slide. Page 1 of 2 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 HS800 Instructions for use v1 06.03.2015 Technique continued: stain is the decolourising step. Over- 6. Wash thoroughly with tap water. decolourising will lead to an erroneous result where 7. Counterstain with Safranin O (HS720); Neutral Gram-positive cells may stain pink to red indicating Red (HS570); or Carbol Fuchsin (HS160) for a Gram-negative result, and under-decolourising up to 1 minute will lead to an erroneous result where Gram- 8. Wash, air dry and examine. 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. 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. Additional Information The thickness of the smear used in the Gram stain will affect the result of the stain. The step that is most crucial in affecting the outcome of the European conformity according to the "IN-VITRO DIAGNOSTIC MEDICAL DEVICES DIRECTIVE 98/79/EC, ANNEX III". Manufactured by TCS Biosciences Ltd. Page 2 of 2 IFU HS800 06.03.2015 V1 .