Grams Stain-Kit K001

Intended Use
Grams Stain Kit is used for differentiation of bacteria on the basis of their gram nature.
Composition**
Ingredients
Gram's Crystal Violet (S012)(Solution A) -
Crystal Violet 2.000 gm
Ethyl alcohol,95% 20.000 ml
Gram's Crystal Violet (S012)(Solution B) -
Ammonium oxalate 0.800 gm
Distilled Water 80.000 ml
Solution A and B are mixed and stored for 24 hours before use.The resulting stain is stable.
Gram's Decolourizer(S032) -
Ethyl alcohol, 95% 50.0 ml
Acetone 50.0 ml
Gram's Iodine(S013) -
Iodine 1.000 gm
Potassium iodide 2.000 gm
Distilled water 300.000 ml
Safranin,0.5% w/v(S027) -
Safranin O 0.500 gm
Ethyl alcohol, 95% 100.000 ml
**Formula adjusted, standardized to suit performance parameters
Directions
1)Prepare a thin smear on clear, dry glass slide.
2)Allow it to air dry and fix by gentle heat.
3)Flood with Gram's Crystal Violet (S012) for 1 minute. (If over staining results in improper decolourization of known gram-
negative organisms,use less crystal violet).
4)Drain the stain.
5)Flood the smear with Gram’s Iodine (S013). Allow it to remain for 1 minute.
6)Decolourize with Gram's Decolourizer (S032) until the blue dye no longer flows from the smear.
7)Wash with tap water.
8)Counter stain with 0.5% w/v Safranin (S027). Allow it to remain for 1 minute.
9)Wash with water.
10)Allow the slide to air dry or blot dry between sheets of clean bibulous paper and examine under oil immersion objective.

Principle And Interpretation

The Gram stain is a differential staining technique most widely applied in all microbiology disciplines laboratories. It is one
of the most important criteria in any identification scheme for all types of bacterial isolates. Different mechanisms have been
proposed to explain the gram reaction. There are many physiological differences between gram-positive and gram-negative
cell walls (1). Ever since Christian Gram has discovered Gram staining, this process has been extensively investigated and
redefined. In practice,a thin smear of bacterial cells is stained with crystal violet, then treated with an iodine containing
mordant to increase the binding of primary stain (2). A decolourizing solution of alcohol or acetone is used to remove the
crystal violet from cells which bind it weakly and then the counterstain (like safranin) is used to provide a colour contrast in
those cells that are decolourized.

Please refer disclaimer Overleaf.

 HiMedia Laboratories Technical Data

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50–90% of cell envelope), and as a result are
stained purple by crystal violet, whereas gram-negative bacteria have a thinner layer (10% of cell envelope), so do not retain
the purple stain and are counter-stained pink by safranin.In a properly stained smear by gram staining procedure, the gram-
positive bacteria appear blue to purple and gram negative cells appear pink to red.

Type of specimen
Clinical samples - Blood, urine, CSF, pus, wounds, lesions, body tissues, sputum etc. ; food & dairy samples ; Water
samples

Specimen Collection and Handling:

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (3, 4).
For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (5, 6).
For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards(7).
After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions :

In Vitro diagnostic Use only. Read the label before opening the container. Wear protective gloves/protective clothing/eye
protection/ face protection. Follow good microbiological lab practices while handling specimens and culture. Standard
precautions as per established guidleines should be followed while handling clincal specimens. Saftey guidelines may be
referred in individual safety data sheets

Limitations :
1. Use results of Gram stains in conjunction with other clinical and laboratory findings. Use additional procedures (e.g.,
special stains, inclusion of selective media, etc.)to confirm findings suggested by gram-stained smears (8).
2. False Gram stain results may be related to inadequately collected specimens or delay in transit.
3. Careful adherence to procedure and interpretive criteria is required for accurate results. Accuracy is highly dependent on
the training and skill of microscopists (9).
4. The sensitivity of Gram stain is 105 cells/ml or 104 if the specimen has been prepared with the cytocentrifuge (10). This
is particularly applicable to the smear of a drop of urine, where an average of the one bacterial cell per field from an
examination of 20 fields correspond to a count of >= 105 cfu/ml.

Performance and Evaluation

Performace of the product is expected when used as per the direction on the label within the expiry period when stored
at recommended temperature.

Quality Control
Microscopic examination
Gram staining is ccarried out and observed under oil immersion lens.
Results
Gram-positive organisms : Violet coloured
Gram-negative organisms : Pinkish red coloured

Storage and Shelf Life

Store between 10- 30°C in tightly closed container and away from bright light. Use before expiry date on label. On opening,
product should be properly stored in dry ventilated area protected from extremes of temperature and sources of ignition.
Seal the container tightly after use.

Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow
established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical
sample must be decontaminated and disposed of in accordance with current laboratory techniques (3,4).

Please refer disclaimer Overleaf.

HiMedia Laboratories Technical Data

Reference
1. Lamanna and Mallette, 1965, Basic Bacteriology, 3rd ed., Williams and Wilkins Co.,Baltimore.
2. Salton, 1964, The Bacterial Cell Wall, Elsevier, Amsterdam.
3. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.
4. Jorgensen,J.H., Pfaller , M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual
of Clinical Microbiology, 11th Edition. Vol. 1.
5. Downes F. P. and Ito K. (Ed.), 2001, Compendium of Methods for the Microbiological Examination of Foods, 4th
ed., APHA, Washington, D.C.
6.Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed.,
APHA Inc., Washington, D.C.
7. Rice E.W., Baird, R.B., Eaton A. D., Clesceri L. S. (Eds.), 2012, Standard Methods for the Examination of Water and
Wastewater, 22nd ed., APHA, Washington, D.C.
8. Brown,M.S.,and T.C. Wu. 1986. The Gram stain morphology of fungi, mycobacteria, and Pneumocytis carinii.
J.Med .Technol3:495-499.
9. Washington, J.A.1986.Rapid diagnosis by microscopy. Clin.Microbiol. Newsl.8:135-137.
10. Shanhooltzer,C.J.,P. Schaper ,and L.R. Peterson.1982.Concentrated Gram stain smear prepared with a cytospin
centrifuge. J.clin. Microbiol.16:1052-1056

Revision : 02 / 2018

In vitro diagnostic medical

IVD device

CE Marking

30°C Storage temperature

10°C

Do not use if package is

damaged

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Disclaimer :
User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained in
this and other related HiMedia™ publications. The information contained in this publication is based on our research and development
work and is to the best of our knowledge true and accurate. HiMedia™ Laboratories Pvt Ltd reserves the right to make changes to
specifications and information related to the products at any time. Products are not intended for human or animal or therapeutic use but
for laboratory,diagnostic, research or further manufacturing use only, unless otherwise specified. Statements contained herein should not
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