Gram’s Staining for medical students.pptx

this ppt well describes the principle, procedure, modification, usage and limitations of gram's staining.
it is a differential staining method used in bacteriology laboratory

1. Gram’s Staining skill based learning ROMA GOYAL 1

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

3. First Paper on Gram Staining In his paper, Dr. Gram described how he was able to visualize what we now call Staphylococcus , Streptococcus , Bacillus , and Clostridia in various histological sections. Interestingly, Dr. Gram did not actually use safranin as a counter stain in the original procedure (Gram negative cells would be colorless). He instead recommended using Bismarck brown as a counter stain to enable tissue cell nuclei to be visualized. 3

4. Carl Weigert ( 1845-1904) Gram did not use a counterstain in his procedure. It was a few years later, that the German pathologist Carl Weigert (1845-1904) from Frankfurt, added a final step of staining with safranin. 4

5. Traditional Definition of Gram stain A method of staining bacteria using a violet stain. The gram staining characteristics (denoted as positive or negative). A heat fixed bacterial smear is stained with crystal violet (methyl violet), treated with 3% iodine/potassium iodide solution, washed with alcohol and counterstained. The method differentiates bacteria into two main classes, gram-positive and gram-negative. 5

6. The Cell walls differ… 6

7. Gram staining observation Basic Principle in Koch’s postulations The first of Koch’s postulate that the suspected the organism should always be found in association with the disease . 7

8. Requirements for Gram staining technique Glass slides (25 by 75 mm), frosted ends desirable b. 0.85% Nacl, sterile c. Pasteur pipettes and wood applicator sticks, sterile d. Microbiological loops, inoculating needles e. Supplies for disposal of biological waste, including “sharps” f. Bunsen burner g. Immersion oil 8

9. Poor quality of slides Can be corrected Use of glass slides that have not been pre cleaned or degreased ? NOTE : Storing slides in a jar with 95% ethanol will ensure clean slides. Drain excess alcohol or flame slide before use. 9

10. Four Major Steps in Gram Staining There are four basic steps of the Gram stain, which include applying a primary stain (crystal violet)or Methyl violet to a heat-fixed smear of a bacterial culture, followed by the addition of a mordant (Gram's iodine), rapid decolorization with alcohol or acetone, and counterstaining with Safranin or basic fuchsin. 10

11. Organizing the Staining Bottles 11

12. Making a Smear First prepare your slide. You do this by placing bacteria on a slide in a drop of normal saline, allowing them to dry and then heat fixing them. Heating the slide kills the bacteria and makes sure that the bacteria a stuck to the slide and wont wash away during the staining procedure 12

13. Correct preparation Smear preparation: Proper smear preparation should produce a monolayer of organisms sufficiently dense for easy visualization but thin enough to reveal characteristic morphological characteristics. Use clean, new glass slides. NOTE: When using the same pipette or swab, always inoculate culture media first 13

14. Method of Picking material from Agar plates Wrong Right 14

15. Making Multiple smears in same slide – conserve resources Making multiple smears make the optimal use of the slide. Reduces the economic costs and saves the technical time. 15

16. Steps in Gram Staining Procedure- Follow the Clock 1 On a rack, flood with filtered crystal violet ( Methyl violet )   1 M inute. 2 Wash briefly in water to remove excess crystal violet 3.   Flood with Gram’s iodine 1 M inute. 4.   Wash briefly in water, do not let the section dry out. 5.   Decolourise with acetone until the moving dye front has passed the lower edge of the section 6.   Wash immediately in tap water 7 .   Counterstain with safranin 30 seconds     16

17. Proceed in organized Fashion 17

18. Step 1 Dr.T.V.Rao MD 18

19. Step 2 Dr.T.V.Rao MD 19

20. Step 3 Dr.T.V.Rao MD 20

21. Step 4 Dr.T.V.Rao MD 21

22. Step 5 Dr.T.V.Rao MD 22

23. Acetone used with Caution Acetone is a more rapid decolorizes than alcohol and must be used with some care. Excessive decolorization turns Gram positive appear as Gram negative 23

24. Which alcohol is better Several alcohols have been studied, and it has been reported that the more complex the alcohol, the slower the decolorization action. As the carbon chain lengthens , decolorization is slower. Kisskalt (84) found decolorization power decreasing in the following order: methyl, ethyl, propyl, butyl, and amyl alcohol. Conn found a mixture of equal parts of methyl and isopropyl alcohols to have very similar decolorization properties to ethyl alcohol . In practice, however, no known advantage can be gained by substituting the higher alcohols for ethyl alcohol. 24

25. Step 6 Dr.T.V.Rao MD 25

26. Step 7 Dr.T.V.Rao MD 26

27. Caring the stained slide After the counterstain has been rinsed off, the slide is placed between some absorbent paper and the excess water gently blotted off . Care must be taken not to rub the slide with the blotting paper because this would remove the adhering bacteria. 27

28. Optimal use of Microscopy Gram stained preparations have to be observed with bright-field optics. Phase-contrast microscopy does not allow the recognition of true colours . Gram-positive bacteria may be seen under phase-contrast as red cells. Using bright-field optics, Gram-positive cells are purple or blue and Gram-negative pink due to counter stain with Safranin.. 28

29. QUALITY CONTROL The Gram ’ s stain reagents should be tested using known gram-positive and gram-negative organisms each day of use. 1) Gram-positive control – Staphylococcus aureus ATCC 25923 2) Gram-negative control – Escherichia coli ATCC 25922. B Do not interpret test unless the controls yield expected results.

30. Report as follows 1 If no microorganisms are seen in a smear of a clinical specimen, report “No microorganisms seen.” 2 . If microorganisms are seen, report relative numbers and Describe morphology. Observe predominant shapes of microorganisms 30

31. Gram Staining – Gram +’ve 31

32. Gram Staining – Gram -’ve 32

33. Value of Direct Smears Guide the physician on initial choice of antibiotic, pending results of culture and sensitivity. Judge specimen quality. Contribute to selection of culture media, especially with mixed flora. Provide internal quality control when direct smear results are compared to culture results. 33

34. Nature of Morphology in guides early Diagnosis in uncommon diseases 34

35. Limitations of Gram’s Staining We know that Gram positivity is restricted almost exclusively to the bacteria, with only a few other groups , such as the yeasts, exhibiting this reaction . 35

36. Better Understanding of Gram’s Staining We know that the Gram stain is not an all-or-nothing phenomenon , but that quantitative variations in Gram-positivity exist between different species, and within the same species during different parts of the growth cycle or under different environmental conditions . We know that only intact cells are Gram-positive, so that cells which are even gently broken become Gram-negative . We know that bacterial protoplasts, devoid of cell wall, are still Gram-positive, indicating that it is probably the semipermeable membrane which is somehow involved in the reaction . 36

37. Stains Several Fungi 37

38. Streptococcus pneumonia 38

39. Streptococcus pneumonia in Sputum 39

40. Gram stain of Neisseria gonorrhoea, 40

41. Nocardia spp seen in Gram Staining 41

42. Gram Stained Actinomyctes spp 42

43. Common errors in Staining procedure Excessive heat during fixation Low concentration of crystal violet Excessive washing between steps Insufficient iodine exposure Prolonged decolourization Excessive counterstaining 43

44. Gram stain results may not correlated with culture results Gram stain-positive, culture-negative specimens may be the result of contamination of reagents and other supplies, presence of Antimicrobial agents, or failure of organisms to grow under usual Culture conditions (media, atmosphere, etc.) Presence of anaerobic microorganism 44

45. Artifacts in Gram Staining Gram stain reagents (Crystal Violet, Iodine, Safranin and Neutral Red Dirty glass slides Contaminated water used to rinse slides When investigating non-viable organisms seen in Gram stains it is always wise to eliminate every potential source. 45

46. Gram staining not a fool proof procedure Gram’s staining method is not without its problems.  It is , complicated, and prone to operator error.   The method also requires a large number of cells However, it  is also central to phenotypic microbial identification techniques.  46

47. Gram variable observations in Gram staining The Gram staining procedure does not always give clear-cut results. Some organisms are Gram-variable and may appear either Gram-negative or Gram-positive according to the conditions. With these types of organisms, Gram-positive and Gram-negative cells may be present within the same preparation 47

48. Why Gram Variability ? Some Gram-positive bacteria appear Gram-negative when they have reached a certain age, varying from a few hours to a few days. On the other hand, some Gram-negative bacteria may become Gram-positive in older cultures. For this reason it is strongly recommended to use very young cultures for the staining procedure, after growth has become just visible. 48

49. Overcoming in Gram Variable Observations I t is necessary that it is stained at two or three different ages (very young cultures should be used). If an organism changes from positive to negative or vice versa during its growth cycle, this change should be recorded with a statement as to the age of the culture when the change was first observed. In case a Gram-variable reaction is observed it is also good to check the purity of the culture. 49

50. Gram Staining appearance differs.. The genera Actinomyctes, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these Gram-positive cells. The staining of these organisms result in an uneven or granular appearance 50

51. Interpret Gram Staining with Clinical Picture and other Investigations Nevertheless, Gram's stain findings can be equivocal and, therefore, must be assessed carefully in light of the clinical picture. 51

52. Modification in Gram staining methods ? Since the original procedure of Gram, many variations of the Gram staining technique have been published. Some of them have improved the method, others include some minor technical variants of no value. Bartholomew (1962) has pointed out that each variation in the Gram staining procedure has a definite limit to its acceptability. 52

53. Modifications -Report with caution Any final result is the outcome of the interaction of all of the possible variables. All modified methods to be practised with caution should suit to the laboratory, and quality control checks. 53

54. Hucker and Conn's recommendation There is no gram procedure which can be referred to as the best for all laboratories and for all situations. It is recommended that the young microbiologists adopt at least two of the well-accepted methods, practice them until he is familiar with their characteristics , 54

55. Better Profienncy with quality controls Use controls of organisms with known gram reactions is excellent counsel. This plan will give much better results than constant change of methods in the hope of finding one that is foolproof 55

56. GRAM STAINING It's a mystery Although it may seem strange, the reason why bacteria with these two major types of bacteria cell walls react differently with Gram's stain appears to be unconnected with the wall structure itself. The exact mechanism of the staining reaction is not fully understood, however, this does not detract from its usefulness. 56

57. Words of Wisdom Hans Christian Gram I am aware that as yet it is very defective and imperfect 57

58. Creating Library of Gram Stains Drain or gently blot excess oil For slide libraries and teaching collections that will be stored for longer periods, immersion oil can be removed with xylene solution and the slides can be cover slipped using Per mount to prevent fading. Dr.T.V.Rao MD 58

59. Gram staining continues to be Most Rapid test. Even new molecular methodologies typically take hours rather than minutes. " This simple staining procedure remains the most useful test performed in the microbiology lab. Results from a Gram's stain can tell volumes about an infection within 15 minutes of a specimen's arrival in the lab, while most other microbiology results require 24 hours or more. 59

60. 60 THANK YOU