Gram Staining Skill Based Learning

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Gram Staining Skill Based Learning

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Gram’s Staining skill based learning :

Gram’s Staining skill based learning Dr.T.V.Rao. MD Dr.T.V.Rao MD 1

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 2

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 3

Carl Weigert (1845-1904):

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. Dr.T.V.Rao MD 4

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 5

The Cell walls differ…:

The Cell walls differ… Dr.T.V.Rao MD 6

Gram Positive Structure:

Gram Positive Structure Peptidoglycan Cell Wall Cytoplasm Cytoplasmic Membrane Cell Section Enlargement = Within the peptidoglycan stand are teichoic acids and polysaccharides

Gram Negative Structure:

Gram Negative Structure Cell Wall Cytoplasm Cytoplasmic Membrane Peptidoglycan Outer Membrane Section Enlargement = Cell The outer membrane contains Porin trimers, O antigens and Lipopolysaccharides. There is also a space between the Cytoplasmic Membrane and the Outer Membrane which is known as the Periplasmic Space

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 9

Requirements for Gram staining technique:

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 Dr.T.V.Rao MD 10

Poor quality of slides Can be corrected:

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

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 the slide kills the bacteria and makes sure that the bacteria a stuck to the slide and wont wash away during the staining procedure 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 Picking material from Agar plates Wrong Right:

Method of Picking material from Agar plates Wrong Right Dr.T.V.Rao MD 16

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 17

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 until the moving dye front has passed the lower edge of the section 6.   Wash immediately in tap water 7 .   Counterstain with safranin 15 seconds Dr.T.V.Rao MD 18

Proceed in organized Fashion:

Proceed in organized Fashion Dr.T.V.Rao MD 19

Step 1:

Step 1 Dr.T.V.Rao MD 20

Step 2:

Step 2 Dr.T.V.Rao MD 21

Step 3:

Step 3 Dr.T.V.Rao MD 22

Step 4:

Step 4 Dr.T.V.Rao MD 23

Step 5:

Step 5 Dr.T.V.Rao MD 24

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 25

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. 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. Dr.T.V.Rao MD 26

Step 6:

Step 6 Dr.T.V.Rao MD 27

Step 7:

Step 7 Dr.T.V.Rao MD 28

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 29

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 30

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 31

Gram Staining – Gram +’ve:

Gram Staining – Gram +’ve Dr.T.V.Rao MD 32

Gram Staining – Gram -’ve:

Gram Staining – Gram -’ve Dr.T.V.Rao MD 33

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 34

Nature of Morphology in guides early Diagnosis in uncommon diseases :

Nature of Morphology in guides early Diagnosis in uncommon diseases Dr.T.V.Rao MD 35

Limitations of Gram’s Staining:

Limitations of Gram’s Staining W e 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 36

Better Understanding of Gram’s Staining:

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 . Dr.T.V.Rao MD 37

Stains Several Fungi:

Stains Several Fungi Dr.T.V.Rao MD 38

Streptococcus pneumonia:

Streptococcus pneumonia Dr.T.V.Rao MD 39

Streptococcus pneumonia in Sputum :

Streptococcus pneumonia in Sputum Dr.T.V.Rao MD 40

Gram stain of Neisseria gonorrhoea, :

Gram stain of Neisseria gonorrhoea, Dr.T.V.Rao MD 41

Nocardia spp seen in Gram Staining:

Nocardia spp seen in Gram Staining Dr.T.V.Rao MD 42

Gram Stained Actinomyctes spp:

Gram Stained Actinomyctes spp Dr.T.V.Rao MD 43

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 44

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 microorganism Dr.T.V.Rao MD 45

Artifacts in Gram Staining:

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. Dr.T.V.Rao MD 46

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 cells However, it  is also central to phenotypic microbial identification techniques. Dr.T.V.Rao MD 47

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 48

Why Gram Variability?:

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. Dr.T.V.Rao MD 49

Overcoming in Gram Variable Observations:

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. Dr.T.V.Rao MD 50

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 51

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 52

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. 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 53

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 54

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 55

Better Profienncy with quality controls:

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 Dr.T.V.Rao MD 56

GRAM STAINING :

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. Dr.T.V.Rao MD 57

Words of Wisdom Hans Christian Gram :

Words of Wisdom Hans Christian Gram I am aware that as yet it is very defective and imperfect Dr.T.V.Rao MD 58

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 59

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 60

Slide 61:

Created by Dr.T.V.Rao MD for Medical and Paramedical students in the Developing World Email doctortvrao@gmail.com Dr.T.V.Rao MD 61

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