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Gram Staining inClinical Microbiology              Dr.T.V.Rao. MD         Professor of Microbiology Travancore Medical Col...
The Guest Lecture presented by Dr.T.V.Rao MD                     at         • Tenth National Workshop on    • “Simple Diag...
Working at Mansa General Hospital Mansa                Republic of ZambiaTaught many lessons, to understand Infection and ...
Hans Christian Gram                 • The Gram stain was                   devised by the Danish                   physici...
First Paper on Gram Staining• In his paper, Dr. Gram described how he was  able to visualize what we now call  Staphylococ...
Carl Weigert (1845-1904)                  • German                    pathologist Carl                    Weigert (1845-  ...
Traditional Definition of Gram stain• A method of staining bacteria using a violet  stain. The gram staining characteristi...
The Cell walls differ…         Dr.T.V.Rao MD   8
Gram Positive should not be Mistaken• In the Gram Stain technique, two  positively charged dyes are used:  crystal violet ...
Gram staining observationBasic Principle in Koch’s postulations• The first of  Koch’s postulate  that the  suspected the  ...
Poor quality of slides            Can be corrected• Use of glass slides  that have not  been pre cleaned  or degreased ? N...
Four Major Steps in Gram Staining• There are four basic steps of the Gram  stain, which include applying a primary  stain ...
Organizing the Staining Bottles             Dr.T.V.Rao MD        13
Making a Smear• First prepare your  slide. You do this  by placing bacteria  on a slide in a drop  of water, allowing  the...
Correct preparation• Smear preparation: Proper smear  preparation should produce a monolayer of  organisms sufficiently de...
Method of smearing the Material     Wrong                    Right              Dr.T.V.Rao MD           16
Using Methanol is it Better than        Heat Fixation ?• Fix the smear with  95% Methanol• Which will help in  prevention ...
Making Multiple smears in same slide –         conserve resources• Making multiple  smears make the  optimal use of the  s...
Steps in Gram Staining Procedure-                Follow the Clock• 1 On   a rack, flood with filtered crystal violet    ( ...
Proceed in organized Fashion            Dr.T.V.Rao MD      20
Step 1 Dr.T.V.Rao MD   21
Step 2 Dr.T.V.Rao MD   22
Step 3 Dr.T.V.Rao MD   23
Step 4 Dr.T.V.Rao MD   24
Step 5 Dr.T.V.Rao MD   25
How long you keep Iodine in the        Laboratory ???• The Gram’s Iodine we make in  the laboratory from basic  chemicals•...
Most Critical Step in Gram staining                        • The most critical                          step of gram stain...
Acetone used with Caution• Acetone is a more  rapid decolorizes  than alcohol and  must be used with  some care.• Excessiv...
Which alcohol is better• Several alcohols have been studied, and  it has been reported that the more  complex the alcohol,...
Step 6 Dr.T.V.Rao MD   30
Which counterstain is better• Some bacteria which  are poorly stained by  Safranin, such  as Hemophilus  spp., Legionella ...
Step 7 Dr.T.V.Rao MD   32
Caring the stained slideAfter the counterstain has been rinsed off, the slide is placed between some absorbent paper and t...
Gram staining depends on• Includes culture age, media, incubation  atmosphere, staining methods, . Similar  considerations...
How the Gram Stain Work• So how does it work? Gram didnt know - he  simply worked empirically. We now know that the  Gram ...
Optimal use of Microscopy                   • Gram stained preparations                     have to be observed with      ...
Report as follows• 1 If no microorganisms are seen in a  smear of a clinical specimen, report  “No microorganisms seen.”• ...
A gram stained bacterial suspension containing amixture of Gram negative bacilli, and Gram positive  cocci arranged in bun...
A true Gram Negative staining            Dr.T.V.Rao MD       39
Value of Direct Smears• Guide the physician on initial choice of  antibiotic, pending results of culture and  sensitivity....
Staining depends on Structural         Integrity of Cell Wall• We know that only intact cells are Gram-  positive, so that...
Nature of Morphology guides early Diagnosis           in uncommon diseases                   Dr.T.V.Rao MD              42
Identify• A young patient  presented with  foul smelling  purulent  discharge since 2  days on  observation by  Gram stain...
Gram stain of Neisseria gonorrhoeae,
Observe Spores may appear asGram negative and Gram positive
Burkholderia pseudomallei is a gram-negative  bacilli with a “safetypin” appearance on           microscopic examination  ...
Limitations of Gram’s Staining                      • We know that Gram                        positivity is              ...
Better Understanding of Gram’s            Staining• We should know that the Gram stain is  not an all-or-nothing phenomeno...
Stains Several Fungi        Dr.T.V.Rao MD   49
Streptococcus pneumonia          Dr.T.V.Rao MD   50
Streptococcus pneumonia in Sputum              Dr.T.V.Rao MD         51
Nocardia spp seen in Gram Staining               Dr.T.V.Rao MD         52
Gram Stained Actinomyctes spp            Dr.T.V.Rao MD       53
Faulty Gram stain reactions• It is possible to report as " Gram-  negative" if the gram-positive bacteria  are old, dead, ...
Common errors in Staining            procedure• Excessive heat during  fixation• Low concentration of  crystal violet• Exc...
Gram stain results may not correlated        with culture results• Gram stain-positive, culture-negative  specimens may be...
Artifacts in Gram Staining• Gram stain  reagents Crystal  Violet, Iodine ?,  Safranin,  contaminated.• Dirty glass slides•...
Biochemical Tests in Identification• KOH string test may be  used as a confirmatory  test for the Gram Stain  (Powers, 199...
Gram staining not a fool proof         procedure                     • Gram’s staining method                       is not...
Gram variable observations in Gram              staining• The Gram staining procedure does not  always give clear-cut resu...
Overcoming in Gram Variable        Observations• It is necessary that it is stained  at two or three different ages  (very...
Gram Staining appearance differs..The genera Actinomyctes,Arthobacter,Corynebacterium,Mycobacterium, andPropionibacterium ...
QUALITY CONTROL• Check appearance of reagents daily• If crystal violet has precipitate or crystal  sediment, refilter befo...
QUALITY CONTROL• Daily and when a new  lot is used, prepare a  smear of Escherichia  coli (ATCC 25922) and  Staphylococcus...
Interpret Gram Staining with Clinical   Picture and other Investigations• Nevertheless,  Grams stain  findings can be  equ...
Modification in Gram staining             methods ?• Since the original procedure of  Gram, many variations of the  Gram s...
Modifications -Report with caution• Any final result is the  outcome of the  interaction of all of  the possible  variable...
Is it wise to adopt different Gram         staining procedure • Bartholomew (1962) has    pointed out that eachvariation i...
Hucker and Conns         recommendation• There is no gram procedure which  can be referred to as the best for all  laborat...
Words of WisdomHans Christian Gram                • I am aware                  that as yet it                  is very   ...
Creating Library of Gram Stains  Drain or gently blot      excess oilFor slide libraries and teaching collections that wil...
Best of References        You can read on line….• A monograph of  gram-stained  preparations of  clinical Specimens• By Li...
Gram staining continues to be         Most Rapid test.• Even new molecular methodologies  typically take hours rather than...
Gram’s Staining               A Mystery• The exact  mechanism of the  staining reaction is  not fully  understood,  howeve...
Dr.T.V.Rao MD   75
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Gram staining and Clinical applications

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Gram staining and Clinical applications

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Gram staining and Clinical applications

  1. 1. Gram Staining inClinical Microbiology Dr.T.V.Rao. MD Professor of Microbiology Travancore Medical College, Kollam. Kerala Dr.T.V.Rao MD 1
  2. 2. The Guest Lecture presented by Dr.T.V.Rao MD at • Tenth National Workshop on • “Simple Diagnostic Methods in Clinical Microbiology” • 29th November to 3rd December 2011 • • Department of Microbiology• Jawaharlal Institute of Postgraduate Medical Education & Research, Pondicherry • Dr.T.V.Rao MD 2
  3. 3. Working at Mansa General Hospital Mansa Republic of ZambiaTaught many lessons, to understand Infection and Ignorance are important causes of Morbidity and Mortality Dr.T.V.Rao MD 3
  4. 4. 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. Dr.T.V.Rao MD 4
  5. 5. 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. Dr.T.V.Rao MD 5
  6. 6. Carl Weigert (1845-1904) • German pathologist Carl Weigert (1845- 1904) from Frankfurt, added a final step of staining with safranin. 6 Dr.T.V.Rao MD
  7. 7. 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. Dr.T.V.Rao MD 7
  8. 8. The Cell walls differ… Dr.T.V.Rao MD 8
  9. 9. Gram Positive should not be Mistaken• In the Gram Stain technique, two positively charged dyes are used: crystal violet and safranin. The use of the designation “gram-positive” should not be confused with the concept of staining cells with a simple stain that has a positive charge. Dr.T.V.Rao MD 9
  10. 10. Gram staining observationBasic 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. Dr.T.V.Rao MD 10
  11. 11. 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. Dr.T.V.Rao MD 11
  12. 12. 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 (Grams iodine), rapid decolorization with alcohol or acetone, and counterstaining with Safranin or basic fuchsin. Dr.T.V.Rao MD 12
  13. 13. Organizing the Staining Bottles Dr.T.V.Rao MD 13
  14. 14. Making a Smear• First prepare your slide. You do this by placing bacteria on a slide in a drop of water, allowing them to dry and then heat fixing them. Heating Dr.T.V.Rao MD 14
  15. 15. 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 Dr.T.V.Rao MD 15
  16. 16. Method of smearing the Material Wrong Right Dr.T.V.Rao MD 16
  17. 17. Using Methanol is it Better than Heat Fixation ?• Fix the smear with 95% Methanol• Which will help in prevention of distortion of cells• Helpful in Microscopic observation of CSF and Urine Dr.T.V.Rao MD 17
  18. 18. 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. Dr.T.V.Rao MD 18
  19. 19. Steps in Gram Staining Procedure- Follow the Clock• 1 On a rack, flood with filtered crystal violet ( Methyl violet ) 10 sec 2 Wash briefly in water to remove excess crystal violet• 3. Flood with Gram’s iodine 10 sec• 4. Wash briefly in water, do not let the section dry out.• 5. Decolourise with acetone for few seconds <6 seconds until the moving dye front has passed the lower edge of the section• 6. Wash immediately in tap water• 7. Counterstain with safranin for 15 seconds.. Dr.T.V.Rao MD 19
  20. 20. Proceed in organized Fashion Dr.T.V.Rao MD 20
  21. 21. Step 1 Dr.T.V.Rao MD 21
  22. 22. Step 2 Dr.T.V.Rao MD 22
  23. 23. Step 3 Dr.T.V.Rao MD 23
  24. 24. Step 4 Dr.T.V.Rao MD 24
  25. 25. Step 5 Dr.T.V.Rao MD 25
  26. 26. How long you keep Iodine in the Laboratory ???• The Gram’s Iodine we make in the laboratory from basic chemicals• How long we can use it ?• Why we have to make frequently ? Dr.T.V.Rao MD 26
  27. 27. Most Critical Step in Gram staining • The most critical step of gram staining is the decolorization step as crystal violet stain will be removed from both G+ve & G-ve cells if the decolorizing agent(e.g alcohol ) is left on too long. Dr.T.V.Rao MD 27
  28. 28. 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 Dr.T.V.Rao MD 28
  29. 29. 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. Conn found in practice, however, no known advantage can be gained by substituting the higher alcohols for ethyl alcohol. Dr.T.V.Rao MD 29
  30. 30. Step 6 Dr.T.V.Rao MD 30
  31. 31. Which counterstain is better• Some bacteria which are poorly stained by Safranin, such as Hemophilus spp., Legionella spp. , and some anaerobic bacteria, are readily stained by basic fuchsin, but not Safranin Dr.T.V.Rao MD 31
  32. 32. Step 7 Dr.T.V.Rao MD 32
  33. 33. Caring the stained slideAfter 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. Dr.T.V.Rao MD 33
  34. 34. Gram staining depends on• Includes culture age, media, incubation atmosphere, staining methods, . Similar considerations apply to the interpretation of smears from clinical specimens, and additional factors include different host cell types and possible phagocytosis.• Gram stain permits the separation of all bacteria into two large groups Dr.T.V.Rao MD 34
  35. 35. How the Gram Stain Work• So how does it work? Gram didnt know - he simply worked empirically. We now know that the Gram reaction is based on the structure of the bacterial cell wall.• In Gram-positive bacteria, the dark purple crystal violet stain is retained by the thick layer of peptidoglycan which forms the outer layer of the cell.• In Gram-negative bacteria, the thin peptidoglycan layer in the periplasm does not retain the dark stain, and the pink safranin counterstains the peptidoglycan layer. Dr.T.V.Rao MD 35
  36. 36. 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.. Dr.T.V.Rao MD 36
  37. 37. 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 Dr.T.V.Rao MD 37
  38. 38. A gram stained bacterial suspension containing amixture of Gram negative bacilli, and Gram positive cocci arranged in bunches (Staphylococci spp)
  39. 39. A true Gram Negative staining Dr.T.V.Rao MD 39
  40. 40. 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. Dr.T.V.Rao MD 40
  41. 41. Staining depends on Structural Integrity of Cell Wall• We know that only intact cells are Gram- positive, so that cells which are even gently broken become Gram-negative. Observations suggest 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. Dr.T.V.Rao MD 41
  42. 42. Nature of Morphology guides early Diagnosis in uncommon diseases Dr.T.V.Rao MD 42
  43. 43. Identify• A young patient presented with foul smelling purulent discharge since 2 days on observation by Gram staining
  44. 44. Gram stain of Neisseria gonorrhoeae,
  45. 45. Observe Spores may appear asGram negative and Gram positive
  46. 46. Burkholderia pseudomallei is a gram-negative bacilli with a “safetypin” appearance on microscopic examination Dr.T.V.Rao MD 46
  47. 47. 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. Dr.T.V.Rao MD 47
  48. 48. Better Understanding of Gram’s Staining• We should 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. Dr.T.V.Rao MD 48
  49. 49. Stains Several Fungi Dr.T.V.Rao MD 49
  50. 50. Streptococcus pneumonia Dr.T.V.Rao MD 50
  51. 51. Streptococcus pneumonia in Sputum Dr.T.V.Rao MD 51
  52. 52. Nocardia spp seen in Gram Staining Dr.T.V.Rao MD 52
  53. 53. Gram Stained Actinomyctes spp Dr.T.V.Rao MD 53
  54. 54. Faulty Gram stain reactions• It is possible to report as " Gram- negative" if the gram-positive bacteria are old, dead, or damaged and the cell wall is not intact.• There is no equivalent "false Gram- positive," but a false Gram-positive can occur if the decolorization step is accidentally omitted. Dr.T.V.Rao MD 54
  55. 55. 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 Dr.T.V.Rao MD 55
  56. 56. 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 microorganisms Dr.T.V.Rao MD 56
  57. 57. Artifacts in Gram Staining• Gram stain reagents Crystal Violet, Iodine ?, Safranin, contaminated.• Dirty glass slides• Contaminated water used to rinse slides Dr.T.V.Rao MD 57
  58. 58. Biochemical Tests in Identification• KOH string test may be used as a confirmatory test for the Gram Stain (Powers, 1995, Arthi et al., 2003): The formation of a string (DNA) in 3% KOH indicates that the isolate is a gram- negative organism. Dr.T.V.Rao MD 58
  59. 59. 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 bacteria. Dr.T.V.Rao MD 59
  60. 60. 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 Dr.T.V.Rao MD 60
  61. 61. Overcoming in Gram Variable Observations• It is necessary that it is stained at two or three different ages (very young cultures should be used). In case a Gram-variable reaction is observed it is also good to check the purity of the culture. Dr.T.V.Rao MD 61
  62. 62. Gram Staining appearance differs..The genera Actinomyctes,Arthobacter,Corynebacterium,Mycobacterium, andPropionibacterium have cellwalls particularly sensitive tobreakage during cell division,resulting in Gram-negativestaining of these Gram-positivecells. The staining of theseorganisms result in an unevenor granular appearance Dr.T.V.Rao MD 62
  63. 63. QUALITY CONTROL• Check appearance of reagents daily• If crystal violet has precipitate or crystal sediment, refilter before use even when purchased commercially. NOTE: Some stains, especially basic fuchsin and safranin, can become contaminated. Start with fresh material in a clean bottle.• Evaporation may alter reagent effectiveness; working solutions should be changed regularly Dr.T.V.Rao MD 63
  64. 64. QUALITY CONTROL• Daily and when a new lot is used, prepare a smear of Escherichia coli (ATCC 25922) and Staphylococcus epidermidis (ATCC 12228)or Staphylococcus aureus (ATCC 25923). Fix and stain as described. Dr.T.V.Rao MD 64
  65. 65. Interpret Gram Staining with Clinical Picture and other Investigations• Nevertheless, Grams stain findings can be equivocal and, therefore, must be assessed carefully in light of the clinical picture. Dr.T.V.Rao MD 65
  66. 66. 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. Dr.T.V.Rao MD 66
  67. 67. 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. Dr.T.V.Rao MD 67
  68. 68. Is it wise to adopt different Gram staining procedure • Bartholomew (1962) has pointed out that eachvariation in the Gram stainingprocedure has a definite limit to its acceptability Dr.T.V.Rao MD 68
  69. 69. Hucker and Conns 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, Dr.T.V.Rao MD 69
  70. 70. Words of WisdomHans Christian Gram • I am aware that as yet it is very defective and imperfect Dr.T.V.Rao MD 70
  71. 71. Creating Library of Gram Stains Drain or gently blot excess oilFor 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 71
  72. 72. Best of References You can read on line….• A monograph of gram-stained preparations of clinical Specimens• By Linda M. Marler, Jean A. Siders, Stephen D. Allen (MD.) Dr.T.V.Rao MD 72
  73. 73. 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 Grams stain can tell volumes about an infection within 15 minutes of a specimens arrival in the lab, while most other microbiology results require 24 hours or more. Dr.T.V.Rao MD 73
  74. 74. Gram’s Staining A Mystery• The exact mechanism of the staining reaction is not fully understood, however, this does not detract from its usefulness. Dr.T.V.Rao MD 74
  75. 75. Dr.T.V.Rao MD 75

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