Question

Slide 1: E. coli

Slide 2: S. aureus

Slide 3: Both (mixed)

Describe the results be for both Gram + and Gram - cells if each of the following scenarios occurred during staining:

1.forget to heat fix

2.forget to add iodine

3.switch the primary and counter stains

4. forget to decolorize

5. decolorize for too long

6. decolorize too quickly

7. forget to add safranin

Answer 1

E.coli or Escherichia coli, is a Gram-negative bacteria that normally lives in your intestines. It is also found in the gut of some animals. Most types of E. coli are harmless and help keep your digestive tract healthy. But some strains can cause diarrhea if we eat contaminated food or drink fouled water.

S.aureus or Staphylococcus aureus is a Gram-positive bacteria.they are frequently found in the upper respiratory tract and on the skin.

we have to tell the results if on staining we do the following then what will happen :

1 : forget to heat fix --- it is the first and most important step of gram staining technique. in heat fixing the bacterial smear which is in liquid form is to be semi solidify and not completely dry. this helps in denature of bacterial enzymes,also prevent from autolysis(self eating of cell's parts).this step helps in sticking or pasting of bacteria on the slide. if this step is forgotten the bacterial smear in the next steps would be washed off as in staining, washing.we also not have to completely dry the bacterial smear it will kill the bacteria.for staining gram positive (S.aureus) or negative bacteria (E.coli) or mixture (E.coli and S.aureus) of both it is most important step.

2. forget to add iodine----if we forget to add iodine in the staining, the crystal violet which enters the cell will not be able to form the Crystal Violet Iodine (CVI) complex. iodine is added after staining the smear with crystal violet. after staining it is washed with ethyl alcohol.as E.coli is a gram negative bacteria it will lose the stain as it has thin layer of  peptidoglycan. here iodine is a mordant dye that helps other on fixing it's color.as E.coli is a gram negative(E.coli) bacteria we will get right result but if any gram positive bacteria (S.aureus) is there it will produce incorrect result as on staining further with safranin both will appear orange under microscope and we will have wrong result.and the mixture of both (E.coli and S.aureus) will appear orange color and we will not be able to identify which is which type of bacteria.

3. switch the primary and counter stains--------CRYSTAL VIOLET is the primary stain and SAFRANIN is the counter stain but if both are switched that means CRYSTAL VIOLET becomes counter stain and SAFRANIN becomes primary stain. if this is done we will not be able to differentiate between gram positive bacteria(S.aureus) and gram negative(E.coli) bacteria as in the last step crystal violet is used and it will retain its color as we have no further procedure to wash the stain and both types of bacteria appear blue in color.in case of the mixture of both all bacteria appear blue in color.

4. forget to decolorize-----in staining ethyl alcohol is used , it helps in dehydration of peptidoglyan layer (make it shrink and tighten).The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria. but in gram negative bacteria peptidoglycan layer is thin and get decolorized but if in E.coli (gram-negative bacteria) we forget to decolorize it will appear in blue color under microscope after completing all the steps and we will again produce a wrong result.and in case of S.aureus( gram positive bacteria) they will appear blue in color after last step. and in the mixture of both all will appear blue in color and we can't differentiate between the 2 bacteria.

5. decolorize for too long------it do not affect the gram negative bacteria(E.coli) because they have to lose the stain and this will not affect them as after it they are stained with safranin and will appear orange in color on observing.but if it's gram positive bacteria(S.aureus) decolourizing for too long will result in lose of stain formed by the crystal violet and iodine and at last will produce wrong result.and they appear orange/red colored. in the mixture of both all bacteria will appear orange colored making no difference between them.

6. decolorize too quickly------it will produce wrong result as in case E.coli, gram negative bacteria is decolorize for too short it can still have the stain of crystal violet and iodine and end will produce wrong result. as in case of only E.coli bacterial smear some bacteria may appear blue and as orange in color. and in case if gram positive bacteria(S.aureus) they have to appear blue at end will have no effect except excess staining. and in the mixture many of the bacteria will appear blue and some can appear orange(E.coli) if they have lose the blue stain. so we can't get to a result if all the blue appearing bacteria are gram positive.

7. forget to add safranin--------as it's the last step of staining and safranin is a weakly water soluble dye.it has no effect in gram positive bacteria(S.aureus) as they already have blue color and did not become red/orange on adding safranin. but the gram negative bacteria(E.coli) appear colorless and in case of mixed bacterial smear consisting of both gram positive and gram negative bacteria we are not able to identify if our smear consist of gram negative bacteria or not means gram positive bacteria will appear but gram negative present can't be identified.

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