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HOMEBACTERIOLOGYGram Staining: Principle, Procedure and Results

Gram Staining: Principle, Procedure and Results

February 2, 2015 Acharya Tankeshwar Bacteriology, Staining techniques in Microbiology 46

Gram staining method, the most important procedure in Microbiology, was developed by Danish
physician Hans Christian Gram in 1884. Gram staining is still the cornerstone of bacterial
identification and taxonomic division.

This differential staining procedure separates most bacteria into two groups on the basis of cell wall
composition:
 1. Gram-positive bacteria (thick layer of peptidoglycan-90% of cell wall)- stains purple

2. Gram-negative bacteria (thin layer of peptidoglycan-10% of cell wall and high lipid content) –
stains red/pink

Image 1: Basic classification of Medically

Important Bacteria

Nearly all clinically important bacteria can be detected/visualized using Gram staining method the
only exceptions being those organisms;

1. That exists almost exclusively within host cells i.e. Intracellular bacteria (e.g., Chlamydia)

2. Those that lack a cell wall (e.g., Mycoplasma)

3. Those of insufficient dimensions to be resolved by light microscopy (e.g., Spirochetes)

Contents [hide]

 1 Steps of Gram Staining

 2 Principle of Gram Stain

 3 Procedure of Gram Staining

o 3.1  Smear Preparation
 o 3.2 Gram Staining Procedure/Protocol:

 4 Results:

o 4.1 Reporting Gram smears

 5 Quality Control

Steps of Gram Staining

Classic Gram staining techniques involve the following steps:  

An easy way to remember the steps of the Gram stain

1. Fixation of clinical materials to the surface of the microscope slide either by heating or by
using methanol. (# Methanol fixation preserves the morphology of host cells, as well as
bacteria, and is especially useful for examining bloody specimen material).

2. Application of the primary stain (crystal violet). Crystal violet stains all cells blue/purple

3. Application of mordant: The iodine solution (mordant) is added to form a crystal violet-

iodine (CV-I) complex; all cells continue to appear blue.

4. Decolorization step: The decolorization step distinguishes gram-positive from gram-negative

cells.

5. The organic solvent such as acetone or ethanol extracts the blue dye complex from the lipid-
rich, thin-walled gram-negative bacteria to a greater degree than from the lipid-poor, thick-
walled, gram-positive bacteria.  The gram-negative bacteria appear colorless and gram-
positive bacteria remain blue.

6. Application of counterstain (safranin): The red dye safranin stains the decolorized gram-
negative cells red/pink; the gram-positive bacteria remain blue.

Find information and process for the Preparation of Gram Staining Regent

Principle of Gram Stain

Image 2: Cell wall of Gram Positive and Gram Negative Bacteria

The differences in cell wall composition of Gram-positive and Gram-negative bacteria account for the
Gram staining differences. Gram-positive cell wall contains a thick layer of peptidoglycan with
numerous teichoic acid cross-linking which resists the decolorization.

In aqueous solutions, crystal violet dissociates into CV+ and Cl – ions that penetrate through the wall
and membrane of both Gram-positive and Gram-negative cells. The CV+ interacts with negatively
charged components of bacterial cells, staining the cells purple.

When added, iodine (I- or I3-) interacts with CV+ to form large crystal violet-iodine (CV-I) complexes
within the cytoplasm and outer layers of the cell.

The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the
membranes of both gram-positive and gram-negative bacteria.

The outer membrane of the Gram-negative cell (lipopolysaccharide layer)  is lost from the cell,
leaving the peptidoglycan layer exposed. Gram-negative cells have thin layers of peptidoglycan, one
to three layers deep with a slightly different structure than the peptidoglycan of gram-positive cells.
With ethanol treatment, gram-negative cell walls become leaky and allow the large CV-I complexes
to be washed from the cell.

The highly cross-linked and multi-layered peptidoglycan of the gram-positive cell is dehydrated by

the addition of ethanol. The multi-layered nature of the peptidoglycan along with
the dehydration from the ethanol treatment traps the large CV-I complexes within the cell.

After decolorization, the gram-positive cell remains purple in color, whereas the gram-negative cell
loses the purple color and is only revealed when the counterstain, the positively charged
dye safranin, is added.

Procedure of Gram Staining

 Smear Preparation
Fix material on a slide with methanol or heat. If the slide is heat fixed, allow it to cool to the touch
before applying the stain.

Image 3: Procedure of Gram Staining; note the color

change after each step

Gram Staining Procedure/Protocol:

1. Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent.
Please note that the quality of the smear (too heavy or too light cell concentration) will
affect the Gram Stain results.

2. Wash slide in a gentle and indirect stream of tap water for 2 seconds.

3. Flood slide with the mordant: Gram’s iodine. Wait 1 minute.

4. Wash slide in a gentle and indirect stream of tap water for 2 seconds.

5. Flood slide with decolorizing agent (Acetone-alcohol decolorizer). Wait 10-15 seconds or

add drop by drop to slide until decolorizing agent running from the slide runs clear.

6. Flood slide with a counterstain, safranin. Wait 30 seconds to 1 minute.

7. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent
and then blot dry with absorbent paper.

8. Observe the results of the staining procedure under oil immersion (100x) using a Bright field
microscope.

If you are struggling to remember the staining reagents used in this procedure and their order you
can remember this sentence  “Come  In  And  Stain” i.e. the order is  Crystal
violet,  Iodine,  Alcohol/Acetone and the final one is  Safranin.

Results:

 Gram-negative bacteria will stain pink/red and

 Gram-positive bacteria will stain blue/purple.

You may also Like

 1. Preparation of Gram stain Reagents

2. Differences between Gram-positive and Gram Negative bacteria

3. Gram-Negative Cocci and Coccobacilli of Medical Significance; List of Bacteria and Diseases

4. Importance of Gram Stain in Anaerobic Bacteriology

Reporting Gram smears

The report should include the following information:

Staphylococcus in Gram Stain

 Numbers of bacteria present, whether many, moderate, few, or scanty

 Gram reaction of the bacteria, whether Gram-positive or Gram-negative

 Morphology of the bacteria, whether cocci, diplococci, streptococci, rods, or coccobacilli.

Also, whether the organisms are intracellular.

 Presence and number of pus cells

 Presence of yeast cells and epithelial cells.

Example
A urethral smear report might read: ‘Moderate numbers Gram-negative intracellular diplococci and
many pus cells.’

Quality Control

Always check new batches of stain and reagents for correct staining reactions using a smear
containing known Gram-positive and Gram-negative organisms.

Variations in Gram Staining Results 

Various factors influence the results of Gram staining. Sometime the result might be entirely
different than you have anticipated. Find out about ‘variations in Gram Staining Results in this
post’.

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Related

Types of Staining techniques used in Microbiology and their applicationsAugust 10, 2013In
"Bacteriology"

Differences between Gram positive and Gram Negative bacteriaAugust 11, 2013In "Bacteriology"

Variations in Gram Staining ResultsApril 16, 2013In "Bacteriology"

 GRAM STAINING REACTION

About Acharya Tankeshwar  455 Articles

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working
as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan
Academy of Health Sciences, Nepal. If you want me to write about any posts that you found
confusing/difficult, please mention in the comments below.

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RELATED ARTICLES
Differences between Gram positive and Gram Negative bacteria

Importance of Gram Stain in Anaerobic Bacteriology

Preparation of Gram stain Reagents

46 COMMENTS

1. Anonymous says:

MARCH 30, 2015 AT 12:15 PM

The Note Given Is So Good!But Would You Mind Giving Additional Notes About Bacterial Growth
And Its Classification?

REPLY

2. Ranishree says:

AUGUST 31, 2015 AT 10:51 PM

Nice explanation.it’s really helpful during viva in exam

REPLY

o Dr Obinna says:

NOVEMBER 2, 2019 AT 12:20 AM

Simple and concise good for fast learning

REPLY
 3. Jean says:

SEPTEMBER 17, 2015 AT 3:58 PM

Very good description

REPLY

4. Anonymous says:

SEPTEMBER 29, 2015 AT 8:26 AM

Am honored for the information

REPLY

5. Tim Timothyrhyz says:

OCTOBER 8, 2015 AT 12:21 PM

I love the GrAm staining,its precise but resourceful,thanks

REPLY

6. ISRAEL MUDOMBO says:

OCTOBER 12, 2015 AT 1:53 PM

Nice explanations on Gram staining, will help in coming exams and assignments. thanks

REPLY

7. Anonymous says:

OCTOBER 14, 2015 AT 10:24 AM

Excellent explanation

REPLY

8. srinath kn says:

OCTOBER 19, 2015 AT 10:12 AM

excellent information about gram staining with clear explanation

REPLY
 9. hajra says:

JANUARY 5, 2016 AT 3:30 PM

It’s perfect…………

REPLY

10. Mamta says:

JANUARY 11, 2016 AT 5:26 PM

It is really helpful… Thanks

REPLY

11. Anonymous says:

FEBRUARY 8, 2016 AT 10:25 AM

Great work sir

REPLY

12. Ally Mwaseba says:

MARCH 8, 2016 AT 7:30 AM

Thanks sir

REPLY

13. Sahr Foday Jr says:

MARCH 17, 2016 AT 9:43 AM

Is a very simlpify and perfect note on gram staining

REPLY

14. Anonymous says:

MARCH 22, 2016 AT 9:05 AM

Thank you!

REPLY
 15. Anonymous says:

APRIL 24, 2016 AT 2:53 PM

aim for cell wall staining and principle that is used on cell wall staining

REPLY

16. Dee Tamanii says:

MAY 6, 2016 AT 12:15 AM

its so helpful…thank you…

REPLY

17. Bibek says:

MAY 18, 2016 AT 9:12 AM

it’s really help me..thanks slots sir,

REPLY

18. Anonymous says:

MAY 26, 2016 AT 9:02 AM

Thank you sir

REPLY

19. lameck says:

JUNE 3, 2016 AT 8:26 AM

i appreciate it!

REPLY

20. Edwin Nopher says:

NOVEMBER 1, 2016 AT 3:08 AM

Nice

REPLY
 21. DEEPAK says:

NOVEMBER 10, 2016 AT 4:02 AM

sir plz mention the objectives and procedures step by step,which i can apply it during examinations
….
and thanks a lot for sharing this…

REPLY

22. Brindha says:

DECEMBER 12, 2016 AT 3:18 PM

After gram staining how long can the slide be used for examination under light microscope

REPLY

23. Mohamoud from Somalia says:

JANUARY 19, 2017 AT 12:23 PM

Excellent explanation. it helped me a lot

REPLY

24. dobeguy says:

JANUARY 25, 2017 AT 3:19 PM

very good notes. but Is there any other references concerning gram staining?

REPLY

25. safa mohammed says:

FEBRUARY 2, 2017 AT 4:51 PM

Thank you this is very good notes

REPLY

26. SAB says:

FEBRUARY 12, 2017 AT 2:59 AM

Does gram positive and negative affect in conceiving

REPLY

27. Clive Feremba says:

APRIL 9, 2017 AT 4:28 PM

thanks so much ,,,,,,clear explanation

REPLY

28. Shreya Tiwari says:

MAY 3, 2017 AT 9:52 PM

The language is really very simple and the notes given are very concise and accurate…..

REPLY

29. Christopher anguzu says:

JULY 2, 2017 AT 7:00 AM

It’s nice and clean it can help in when one is stack thanks

REPLY

30. dina pearl says:

JULY 10, 2017 AT 1:54 PM

Its concise & clear , I love it. Thanks a million

REPLY

31. ashokreddy says:

JULY 11, 2017 AT 12:06 PM

THANK YOU SIR SEND YOUR MAIL ID SIR ( ashokreddyap@gmail,com ) for this given on anydoughts
and question i can ask you thank you

REPLY

32. tk says:
AUGUST 23, 2017 AT 12:36 PM

thanks alot

REPLY

33. NAGENDRA PRASAD says:

SEPTEMBER 7, 2017 AT 11:14 AM

Nice explanation of gram staining procedure. Can you provide the process for test of protein in urine
24 hr.

REPLY

34. Sandy Darby says:

OCTOBER 5, 2017 AT 8:24 PM

A blood culture was performed with a gram stain.

The gram stain was reported out the following day as “Light gram positive cocci”.
Two days later, the culture was reported as “Heavy e-coil.
Can gram positive cocci become gram negative rods?

REPLY

o Tankeshwar Acharya says:

OCTOBER 7, 2017 AT 12:27 PM

No, it wont. Possible explanation may be, there is contamination with Gram positive cocci (CONS)
and E.coli is the causative agent. But they gave you result within 24 hours that sound’s strange too.

REPLY

35. Mus'ab says:

NOVEMBER 21, 2017 AT 4:56 PM

Tnx Alot Sir

REPLY

36. kuldeep says:

DECEMBER 1, 2017 AT 5:11 AM

sir i am student and i need to know why the cell wall of the gram get stained
REPLY

37. kuldeep says:

DECEMBER 1, 2017 AT 5:13 AM

your information is to easy its easy to understand

REPLY

38. Steve Cook says:

DECEMBER 9, 2017 AT 3:37 PM

Personally I dislike the use of acetone/alcohol in the decolourisation step. I much prefer 70%
ethanol. The use of acetone, I believe, is too aggressive and means that timing becomes an issue.
This was also an issue for me with the Brown/Brenn modification that had a vogue in the 1960s. I
may, of course, be a little rusty as I last worked full-time in microbiology in 1970 (I was at the Central
Veterinary Laboratory of the UK Ministry of Agriculture Fisheries and Food). I have since taught
elements of microbiology at secondary and tertiary level.

REPLY

39. kareem bukola says:

JANUARY 27, 2018 AT 4:30 PM

Please,I need your help with journals or other materials on listeria monocytogenes isolation from
ready to eat vegetables like carrot,cabbage,tomato,lettuce and cucumber,also step by step
procedures on the process to take,thanks sir

REPLY

40. amankwa mershack says:

APRIL 18, 2018 AT 1:20 AM

gram staininig is quite interesting

REPLY

41. mutama john says:

MAY 15, 2018 AT 6:18 PM

it’s good work done and just appreciate for your effort
REPLY

42. p.ahila says:

JUNE 7, 2018 AT 9:03 PM

sir,thank u for given this notes.i want clear details in every organisms.

REPLY

43. gilbert says:

JULY 3, 2019 AT 6:04 PM

thnx, in need of pdf

REPLY

44. ANGEL SM says:

JULY 2, 2020 AT 4:23 PM

HOW WERE THE CELLULAR STRUCTURES AFTER COUNTER STAINING WITH 70% ETHANOL

REPLY

Do you have any queries? Please leave me in the comments section below. I will be happy to read
your comments and reply.

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