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- Why is it necessary to use a 24-hrold culture in
gram staining ? - What does Gram staining reveal owing to its use in clinical diagnosis?
- Indicate color staining results of gram positive and negative bacteria for every hypothetical scenario.
Modification in Procedure |
Staining results |
1. Crystal violet was replaced with methylene blue. |
G+ G- |
2. Mordant was skipped |
G+ G- |
3. Decolorizing step was omitted |
G+ G- |
4. counterstain was not used? |
G+ G- |
5. Crystal violet and safranin was swapped in position. |
G+ G- |
- What bacterial specimen represent the following: (a) positive simple staining; and (b) negative simple staining viewed under OIO. Provide details such as specimen and bacterial stain used and reference.
Positive Staining
1000 X
Negative Staining
1000 X
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- Indicate color staining results of gram positive and negative bacteria for every hypothetical scenario. Modification in Procedure Staining results 1. Crystal violet was replaced with methylene blue. Gram positive Gram negative 2. Mordant was skipped Gram positive Gram negative 3. Decolorizing step was omitted Gram positive Gram negative 4. counterstain was not used? Gram positive Gram negative 5. Crystal violet and safranin was swapped in position. Gram positive Gram negativeTable 1. Different Staining Techniques Gram Stain Acid Fast Endospore Capsule Flagella 1. Principle 2. Characteristic of Bacteria viewed using this stain. (e.g. bacterial type, strain, or targeting particular bacterial feature/s) 3. Stain/s Used 4. Type of Stain 5. Mechanism of Staining stain used) (of each 6. Flow of MethodExplain why only gram-negative cells undergo decolorization during the gram staining procedure. Cite the purpose of each of the following reagents in a differential staining procedure. Primary stain Mordant Decolorizing Agent Counter stain What might happen if the Gram staining procedure is performed on a culture incubated for a little over a day
- 1.)What is the purpose of a counterstain? 2. What does a mordant do in the Gram stain procedure? Which reagent in the Gram stain is the mordant? 3. True or False? The oil objective should make contact with the oil on the slide. 4. Why is it necessary to let bacterial smears completely air dry before heat fixing? 5.Why should controls be included wherever possible for any staining technique? 6. Why is it necessary to heat the slides while staining for endospores?indicators, key used for/ determines reagents, or key positive result ingredients 1. crystal violet Comments/ additional info Medium/test negative result decolorization is key. Must include +/- controls on gram reaction and size and 2. iodine morfdant Gram stain stains purple stains pink 3. gram's declorizer 4. safranin shape every slide to verify result quadrant streak- isolation of pure culture of bacteria check this for any pigmentation of colonies TSA Eosin methylene blue agar (EMB) 1. sulfur/H2S 2. indole/tryptophasnase 3. motility/flagella 1. blackening of medium 2. red color in added reagent 3. fuzzy appearance of 1. no blackening of medium 2.same color of added multi test medium (3 results reagent (not red) 3. stab is visible an defined with growth 1. ferrous salts 2. Kovac's SIM in one medium) 3. low % agarose medium/stab not visible motility agar Brewer's plate in Anaerobe jar Fluid thioglycollate (FTM) Citrate slant phenol red fermentation broth test media (list the specific…Types of Differential Staining techniques. 1. Gram stain 2. Acid-fast stain Please explain, thank you.
- Why is it important to limit the quantity of cells used to prepare a smear? Mark all that apply: 1. So that cells are not clumped and don't entrap stain creating erroneous results 2. So that the cells are spread out enough that cell morphology can be discerned 3. So that there are small groups of cells clumped together to make them visible 4. So that no contaminants are introduced onto the slide by being entrapped in clumps 5. So that the cells are spread out enough that the arrangement can be observedWhich of the following is/are true regarding the acid-fast stain? (There may be more than one correct answer.) Non-acid-fast microbes appear blue in a completed acid-fast stain. O Acid-fast cells retain the primary dye after treatment with acid-alcohol. O It is used to identify members of the genus Mycobacterium. V Acid-fast cells appear bright pink/red in a completed acid-fast stain. O If cells are acid-fast, they are gram-negative.Select all that apply to a negative stain: 1. involves a washing step 2. cells may be distorted or shrunken 3. uses an acidic or negatively charged dye which stains the background 4. uses multiple dyes in the procedure 5. uses only 1 dye in the procedure 6. involves fixing 7. does not involve fixing 8. cells will not be distorted or shrunken 9. does not involve a washing step 10. can show cell morphology, size, and arrangement 11. uses a basic or positively charged dye which stains the bacterial cells
- 1.why is gram stain considered a differential stain? 2.How do gram positive and gram negative bacteria differ in cellular structure, and how does this contribute to their differential staining properties? 3.How does the age of a culture affect the gram stain reaction? What is an optimum culture age for a valid gram reaction? 4.Which step in the gram stain procedure is most prone to error?If done correctly how might that step affect the end result? 5.what is the function of mordant, and which reagent serves this purpose in the gram stain procedure? 6.List the reagents of the gram star technique in order and their general role in the staining process. 7.In what type of cell, gram -positive or gram-negative , would you find lipopolysaccharide in its cell wall?Why should agar media be completely dissolved before they are dispensed in tubes and plates? What are the bases for pegging the temperature at 1210C for 15-30 minutes during moist heat sterilization and 1800C for two (2) hours using dry heat sterilization? Can you sterilize culture media using dry heat sterilization? Why is that so? You will notice in the videos shown, the cotton plug is not used. What is role of cotton plug in media prep, sterilization and culture of microorganisms? Instead of using cotton plug, plastic screw-cap is used, can you substitute this for the former? Is it technically acceptable in microbiology?1-What microbial characteristics can one ascertain from a simplest stain? 2-A student is directed to make a simple stain of coli with crystal violet, but got mixed up and used safranin instead. How would their observations be different? Would the information obtained be any different? 3-How is the procedure different when taking cells from a solid medium compared to taking cells from a liquid medium? Why is it important that your smear be thin?