Gram staining Gram positive and gram negative bacteria can be distinguished by Gram Staining

Gram Staining Gram-positive and Gram-negative bacteria Principle of Gram stain, Heat fixing, Gram's iodine, Hans Christian gram, Carl Weighert, Smear making, heat fixing, Precautions for Gram staining, Gram staining distinguish bacteria on the basis of their cell wall composition.

1. Gram Staining Prepared by Agrani Paudel BSc Microbiology first year

2. History Gram stain was developed by the Danish physician, Hans Christian Gram , while working in Berlin in 1883. He later published his procedure in 1884. At the time, Dr Gram was studying lung tissue sections from patients who had died of pneumonia.

3. Cont. Carl Weigert (1845-1904) Gram did not use a counterstain in his procedure German pathologist Carl Weigert added final step of staining with safranin

4. Gram Staining It is one of the most important staining method in bacteriology It differentiates bacterial species into two major groups i. Gram positive bacteria ii. Gram negative bacteria Here with the help of gram staining bacteria differs due to difference in cell wall composition

5. Gram positive bacteria Have thick wall of peptidoglycan Peptidoglycan is polymer of sugar amino acids In between cell wall and cell membrane there is membrane teichoic acid Very thin layer of lipopolysaccharide is present

6. Gram negative bacteria Outer membrane of phospholipids Lipopolysaccharides outside of thin peptidoglycan layer Space between outer membrane and the peptidoglycan layer is called the periplasmic space

7. Principle of Gram staining Stain is fixed due to formation of CVI complex All bacteria stained purple Bacteria Some cells will be decolorized while some will retain stain Cells that retain primary stain i.e. Crystal violet are Gram positive And those that retains the colour of counter stain i.e. safranin are Gram negative Stained with crystal violet Addition of Gram’s iodine Addition of decolorizer alcohol or acetone Staining with safranin

8. Difference in action in cell wall For Gram’s Iodine Forms (crystal violet iodine) CV-I complex by binding to primary stain Only in Gram positive cells, CV-I complex binds to the magnesium –ribonucleic acid component of the cell wall forming magnesium- ribonucleic acid- crystal violet iodine (Mg-RNA-CV-I) complex which is more difficult to remove than smaller CV-I complex.

9. For decolorizing agent Functions as lipid solvent and protein dehydrating agent In Gram Positive cells , small amount of lipid content is readily dissolved by the action of alcohol, forming minute cell wall pores which later is closed by alcohol dehydrating effect. So, the larger Mg-RNA-CV-I complex is difficult to remove and hence remains purple. In case of Gram negative cells , outer thick lipid layer is dissolved by alcohol, creating large pores in cell wall that do not close appreciably on dehydration of cell wall proteins. As a result, release of unbound CV-I complex is facilitated leaving these cell unstained

10. Basic steps of gram staining Applying primary stain(crystal violet) or methyl violet to a heat fixed of bacterial culture Addition of mordant(Gram’s iodine) Rapid decolorization with alcohol or acetone Counterstaining with safranin or basic fuchsin

11. Materials and chemicals required Inoculating loop Clean grease free slide Bunsen burner Primary stain- crystal violet Mordant –Gram iodine Decolorizer- alcohol or acetone Counter stain- safranin

12. Procedure Lab table is disinfected applying a disinfectant such as Lysol All essentials materials are arranged in aseptic environment chemical resistant gloves and chemical splash goggles is recommended to be used if the microorganism is highly pathogenic

13. The slide( clean, dry, grease free) is marked where smear is to be prepared step 1: smear making

14. For liquid culture, two loops of liquid bacterial growth is taken in the center of clean slide One drop of distilled water is added at center of slide for solid culture being used Step 1 smear preparation

15. Inoculating loop is sterilized and let it to cool, if it is too hot it may distort microorganisms

16. colony is added in distilled water with inoculating loop

17. loop is sterilized

18. Now smear is spread in a circular motion to about 1cm in diameter It is left for air dry

19. step 2: Heat fixing Step 2 heat fixing slide is warmed by back and froth movement for attachment of microorganisms to the slide

20. step 3 : smear is stained with crystal violet for 60 sec Step 3 smear stained with crystal violet for about 60 s Gram crystal violet stain is applied from an eye-dropper or pipet onto and slightly beyond the smear area on the slide and is allowed to sit for one minute.

21. step 4: rinsed with distilled water Step 4 : rinsed with distilled water Step 4 : rinsed with distilled water slide is tilted on an angle and the slide is gently rinsed with distilled water from a dropper bottle by dropping water above the smear and allowing it to rinse the stain off the smear. It is rinsed until the solution dripping from the slide is clear.

22. step 5 : applying mordant Gram iodine Step 5 Gram’s iodine is added for 60s Gram iodine is applied from an eye-dropper or pipet onto and slightly beyond the smear area on the slide and was allowed to sit for one minute

23. step 6 : Rinse with distilled water Step 6 rinsed with distilled water Step 6 rinsed with distilled water slide is tilted on an angle and the slide is gently rinsed with distilled water from a dropper bottle by dropping water above the smear and allowing it to rinse the stain off the smear. It is rinsed until the solution dripping from the slide is clear.

24. step 7 : decolorized with acetone or ethanol Step 7 : decolorized with alcohol or acetone microbial smears is decolorized with 95% ethyl alcohol by applying drop-wise for approximately 30 seconds or to a tipped slide until no more color runs off.

25. step 8 : rinse with distilled water Step 8 : rinsed with distilled water Decolorization time is determined primarily by the thickness of the smear. It is very important not to over-decolorize. Then it is rinsed with water

26. step 9 : counter stain with safranin Step 9 : counter stain is added for 45 s Gram safranin counter stain is applied for 30–45 seconds. Safranin serves to stain bacterium which are gram-negative that do not hold crystal violet after decolorization

27. step 10 : rinse with distilled water Step 10 : rinsed with distilled water slide is tilted on an angle and the slide is gently rinsed with distilled water from a dropper bottle by dropping water above the smear and allowing it to rinse the stain off the smear. It is rinsed until the solution dripping from the slide is clear.

28. step 11 : slide is gently dried Step 11 : gently dried Step 11 : gently dried

29. step 12 : oil emersion is added above the smear Step 12 oil emersion is added

30. step 13 : observation is done under microscope Step 13 : observed under microscope

31. Escherichia coli Gram negative bacilli

32. Salmonella typhus Gram negative bacilli

33. Shigella dysenteriae Gram negative bacilli

34. Neisseria meningitidis Gram negative cocci

35. Neisseria gonorrhoeae Gram negative cocci

36. Streptococcus pneumoniae in sputum Gram positive cocci

37. Bacillus anthracis Gram positive bacilli

38. Actinomycetes spp Gram positive filamentous

39. Streptococcus mutans Gram positive cocci

40. Precautions Culture selection Fresh and young culture should be used to avoid misleading results because old cultures of gram positive tend to decolorize more rapidly and show Gram negative ness ;lose the ability to retain crystal violet iodine complex It is possible to report Gram negative bacteria if Gram positive bacteria is old , dead ,damaged and the cell wall is not intact

41. Precautions Smear preparation Smear should be thin and uniform to avoid over populated bacteria but sufficiently dense for easy visualization Even smear should be made other wise alcohol will continue to wash violet colour from thick parts of the smear while thin parts being over decolorized

42. Precautions Heat fixing If slide is not completely dried when you pass it through flame organisms will be boiled and destroyed If too much heat fixing is done , organisms may be incinerated and you will see distorted cells and cellular remains If you heat fix too much little , organism may not stick and will wash off the slide in subsequent steps

43. Precautions During decolorization Over decolorization should be avoided, as over treatment washes crystal violet complex from gram positive and they appear to be gram negative Acetone decolorizes faster than alcohol False Gram positive report, if decolorization step is omitted After rinsing counter stain care must be taken not to rub the slide with blotting paper as this could remove the adhering bacteria

44. Be careful Genera Actinomycetes, Arthrobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division resulting in Gram negative staining of these Gram positive cells. Staining of these organisms results in an uneven or granular appearance Mycobacteria  contain mycolic acids and have a high GC content in their DNA. A  Gram stain cannot  penetrate the waxy cell wall. The hydrophobic lipids cause the  Gram stain  to give no  staining  or a variable result.(Acid fast staining)