Gram staining and Clinical uses

Gram Staining in Clinical Microbiology : 

Gram Staining in Clinical Microbiology Dr.T.V.Rao. MD Professor of Microbiology Travancore Medical College, Kollam. Kerala Dr.T.V.Rao MD 1

The Guest Lecture presented by Dr.T.V.Rao MD at : 

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

Working at Mansa General Hospital Mansa Republic of ZambiaTaught many lessons, to understand Infection and Ignorance are important causes of Morbidity and Mortality : 

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

Hans Christian Gram : 

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

First Paper on Gram Staining : 

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

Carl Weigert (1845-1904) : 

Carl Weigert (1845-1904) German pathologist Carl Weigert (1845-1904) from Frankfurt, added a final step of staining with safranin. Dr.T.V.Rao MD 6

Traditional Definition of Gram stain : 

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

The Cell walls differ… : 

The Cell walls differ… Dr.T.V.Rao MD 8

Gram Positive should not be Mistaken : 

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

Gram staining observation Basic Principle in Koch’s postulations : 

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. Dr.T.V.Rao MD 10

Poor quality of slidesCan be corrected : 

Poor quality of slidesCan 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

Four Major Steps in Gram Staining : 

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. Dr.T.V.Rao MD 12

Organizing the Staining Bottles : 

Organizing the Staining Bottles Dr.T.V.Rao MD 13

Making a Smear : 

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

Correct preparation : 

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

Method of smearing the MaterialWrong Right : 

Method of smearing the MaterialWrong Right Dr.T.V.Rao MD 16

Using Methanol is it Better than Heat Fixation ? : 

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

Making Multiple smears in same slide – conserve resources : 

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

Steps in Gram Staining Procedure- Follow the Clock : 

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

Proceed in organized Fashion : 

Proceed in organized Fashion Dr.T.V.Rao MD 20

Step 1 : 

Step 1 Dr.T.V.Rao MD 21

Step 2 : 

Step 2 Dr.T.V.Rao MD 22

Step 3 : 

Step 3 Dr.T.V.Rao MD 23

Step 4 : 

Step 4 Dr.T.V.Rao MD 24

Step 5 : 

Step 5 Dr.T.V.Rao MD 25

How long you keep Iodine in the Laboratory ??? : 

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

Most Critical Step in Gram staining : 

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

Acetone used with Caution : 

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

Which alcohol is better : 

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

Step 6 : 

Step 6 Dr.T.V.Rao MD 30

Which counterstain is better : 

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

Step 7 : 

Step 7 Dr.T.V.Rao MD 32

Caring the stained slide : 

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. Dr.T.V.Rao MD 33

Gram staining depends on : 

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

How the Gram Stain Work : 

How the Gram Stain Work So how does it work? Gram didn't 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

Optimal use of Microscopy : 

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

Report as follows : 

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

A gram stained bacterial suspension containing a mixture of Gram negative bacilli, and Gram positive cocci arranged in bunches (Staphylococci spp) : 

A gram stained bacterial suspension containing a mixture of Gram negative bacilli, and Gram positive cocci arranged in bunches (Staphylococci spp)

A true Gram Negative staining : 

A true Gram Negative staining Dr.T.V.Rao MD 39

Value of Direct Smears : 

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

Staining depends on Staining depends on Structural Integrity of Cell Wall : 

Staining depends on 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

Nature of Morphology guides early Diagnosis in uncommon diseases : 

Nature of Morphology guides early Diagnosis in uncommon diseases Dr.T.V.Rao MD 42

Identify : 

Identify A young patient presented with foul smelling purulent discharge since 2 days on observation by Gram staining

Gram stain of Neisseria gonorrhoeae, : 

Gram stain of Neisseria gonorrhoeae,

Observe Spores may appear as Gram negative and Gram positive : 

Observe Spores may appear as Gram negative and Gram positive

Burkholderia pseudomallei  is a gram-negative bacilli with a “safetypin” appearance on microscopic examination : 

Burkholderia pseudomallei  is a gram-negative bacilli with a “safetypin” appearance on microscopic examination Dr.T.V.Rao MD 46

Limitations of Gram’s Staining : 

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

Better Understanding of Gram’s Staining : 

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

Stains Several Fungi : 

Stains Several Fungi Dr.T.V.Rao MD 49

Streptococcus pneumonia : 

Streptococcus pneumonia Dr.T.V.Rao MD 50

Streptococcus pneumonia in Sputum : 

Streptococcus pneumonia in Sputum Dr.T.V.Rao MD 51

Nocardia spp seen in Gram Staining : 

Nocardia spp seen in Gram Staining Dr.T.V.Rao MD 52

Gram Stained Actinomyctes spp : 

Gram Stained Actinomyctes spp Dr.T.V.Rao MD 53

Faulty Gram stain reactions : 

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

Common errors in Staining procedure : 

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

Gram stain results may not correlated with culture results : 

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

Artifacts in Gram Staining : 

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

Biochemical Tests in Identification : 

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

Gram staining not a fool proof procedure : 

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

Gram variable observations in Gram staining : 

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

Overcoming in Gram Variable Observations : 

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

Gram Staining appearance differs.. : 

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 Dr.T.V.Rao MD 62

QUALITY CONTROL : 

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

QUALITY CONTROL : 

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

Interpret Gram Staining with Clinical Picture and other Investigations : 

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. Dr.T.V.Rao MD 65

Modification in Gram staining methods ? : 

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

Modifications -Report with caution : 

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

Is it wise to adopt different Gram staining procedure : 

Is it wise to adopt different Gram staining procedure Bartholomew (1962) has pointed out that each variation in the Gram staining procedure has a definite limit to its acceptability Dr.T.V.Rao MD 68

Hucker and Conn's recommendation : 

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, Dr.T.V.Rao MD 69

Words of WisdomHans Christian Gram : 

Words of WisdomHans Christian Gram I am aware that as yet it is very defective and imperfect Dr.T.V.Rao MD 70

Creating Library of Gram Stains : 

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 71

Best of ReferencesYou can read on line…. : 

Best of ReferencesYou 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

Gram staining continues to be Most Rapid test. : 

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. Dr.T.V.Rao MD 73

Gram’s Staining A Mystery : 

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

Slide 75: 

Dr.T.V.Rao MD 75