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microbio- lab 1
| Question | Answer |
|---|---|
| a pure culture contains------ | a single type of microorganism |
| pure cultures can be used to study------ | pathogenicity, growth requirements, or other characteristics of a species or strain of bacteria |
| contamination of a pure culture can be introduced by----- | direct contact with any surface including air |
| sterilized object | an object free from all living microorganisms |
| Aseptic Technique Procedure | flame inoculating loop in Bunsen burner until red. flame entire length of wire, remove lid/cap on culture, touch culture and place where you want it. once it touches anything else it cannot touch culture again. flame loop before setting it down. |
| simple vs differential stain | simple stain- uses just one dye differential stain- one which has different reactions with different tissues. |
| best dyes | aniline dyes- can be acidic, neutral, or basic, primarily with bacteria acidic or basic dyes are used |
| methylene blue | basic dye, positively charged cation (MB+)= attracted to negatively charged surface of bacterial cell (charge due to ionization of many of the -COOH groups on polysaccharides and proteins of the cell surface) |
| basic dyes | methylene blue safranin basic fuschia crystal violet |
| basic fusia | used to stain the nucleic acids when human cells are lysed in hypotonic solution |
| safranin and crystal violet | used in gram stain to stain the bacterial wall |
| acidic dyes | anions combine more readily with the cytoplasmic elements |
| Eosin | acidic dye- stain Eosinophils easily in human blood because they contain cytoplasmic granules |
| Neutral dyes | made by combining acidic and basic dyes most useful for differentiation staining of the interior structures of eukaryotic cells |
| blood smears | stained with neutral dye (Wright's stain) can differentiate white blood cells into eosinophils, basophils, and neutrophils. |
| color of gram negative | pink |
| color of gram positive | dark purple |
| most living cells including human tissues and many bacteria are gram ------- | negative |
| Representative Genera of Gram positive | staphylococcus streptococcus enterococcus micrococcus clostridium bacillus corynebacterium |
| Representative Genera of Gram negative | Escherichia klebsiella proteus salmonella pseudomonas Neisseria vibrio |
| what can come out of gram staining? | presumptive identification of most common pathogens. this is especially important in cases where severe side effects may develop if treatment is delayed |
| factors that can affect the accuracy of the gram stain | timing of decolorization too thick freshness of reagents improper fixing of smear changes in organism itself age of microorganisms/acidity of the culture |
| factors that can affect the accuracy of the gram stain: timing of decolorization | the gram positive reaction is a matter of degree rather than and absolute. during distaining procedure timing is critical. decolorizind too long can turn any gram positive organism into one that appears gram negative |
| factors that can affect the accuracy of the gram stain: too thick | a smear that is too thick may prevent good de-colorization |
| factors that can affect the accuracy of the gram stain: freshness of reagents | the outcome is also influenced by the concentration and freshness of the reagents |
| factors that can affect the accuracy of the gram stain: improper fixing of smear | cells disrupted by improper fixing of the smear may loose the gram positive trait |
| factors that can affect the accuracy of the gram stain: changes in organism itself | the test may not give expected results because of the changes in the organism itself. gram positiveness can fail to develop in some organisms unless serum or blood is present |
| factors that can affect the accuracy of the gram stain: age of microorganisms/acidity of the culture | positive reaction can be influenced by age of microorganism or by acidity of the culture. cells which stain gram positive in young cultures may turn gram negative in older cultures. best results are obtained with cultures that are less than 48 hrs old. |
| the gram ------- character can be lost, but the gram ----- character cannot be lost | positive, negative |
| gram ---- cells may appear gram ----- on a properly prepared smear, but gram------ calls never appear gram ---- | positive negative negative positive |
| if a properly prepared slide shows both gram positive and gram negative reaction it is considered | gram positive |
| Gram stain procedure | 1. smear is made 2. smear flooded with crystal violet 3. smear is rinsed and flooded with grams iodine 4. rinsed 5. decolorize smear 6. flooded with counter-stain |
| Gram stain procedure: smear is made | smear is made, air dried, and heat fixed on microscope slide. before staining bacteria must be properly attached or FIXED to microscope slide. bacteria killed in process. application of stain to unfixed bacteria may wash it from slide |
| Gram stain procedure: smear flooded with crystal violet | smear is flooded with crystal violet and allowed to react for one min. at this time both gram positive and gram negative are stained purple with the crystal violet stain |
| Gram stain procedure: smear is rinsed and flooded with grams iodine | after rinsing slide thoroughly, its flooded with gram's iodine. the iodine acts as a MORDANT producing a crystal violet-iodine complex that can more effectively bind the cell surface |
| Gram stain procedure: rinsed | after rinsing the iodine off slide its ready for decolorization |
| Gram stain procedure: decolorize smear | acetone-alcohol mixture is used to decolorize smear. timing is critical. ~5sec to get correct results. too long will decolorize the gram positive bacteria. if done properly gram positive retains crystal violet stain and gram negative will appear colorless |
| Gram stain procedure: flooded with counter-stain | in order to see the gram negative bacteria, a counter stain is used. the smear is flooded with safranin which allows gram negative to appear pink/red. if any of the gram positive has been decolorized it too will appear pink/red |
| gram staining procedure identifies: | differential characteristics in the bacterial cell wall |
| clinically important characteristics shared by gram positive organisms as compared to gram negative | GPB (gram positive bacteria)= generally are susceptible to penicillin and bacteriostatic dyes GPB= generally more sensitive to lysis by complement GPB=more resistant to physical methods of control (heat, disinfectants, etc.) GPB produce exotoxins |
| clinically important characteristics shared by gram positive organisms as compared to gram negative | GNB may produce exotoxins, but always make endotoxins; it is part of their cell wall. makes symptoms of GN infections more severe when the bacteria are killed and broken up GPB tend to be more fastidious than GNB |
| exception to gram staining | some bacteria such as spirochetes(cause syphilis)= too small to be visualized w/ light microscope so they can'y be characterized by gram stain. bacteria such as mycobacterium & nocardia don't stain well & are better using Ziehl-Neelsen acid-fast stain |
| look at drawing od gram negative vs gram positive module 1 pg 6 | |
| KOH test | after stirring for 20 sec if a string forms=gram negative, no string = gram positive. the cell wall of GNB is easily disrupted when exposed to dilute base. when disrupted, relatively unfragmented threads of DNA are released from bacteria and= thin string |
| shapes of bacteris | caccus- circular bacillus- oval spirilloun- squiggly vibrius-cresent moon |
| organization of bacterium | diplo-two next to each other tetrad-four in a square staphylo-9 in a square strepto-a line of bacteria |
| Note how the bacteria would appear: Acetone-alcohol step was omitted | Gram Positive: will retain crystal violet stain Gram Negative: will retain crystal violet stain |
| Note how the bacteria would appear: Iodine step is omitted | Gram Positive: no crystal violet iodine, complex-more easily wash out, the crystal violet stain turning it pink Gram Negative: will appear pink |
| Note how the bacteria would appear: acetone-alcohol sits 90 seconds | Gram Positive: decolorization of bacteria-will turn pink Gram Negative: will appear pink |
| Note how the bacteria would appear: safranin step is omitted | Gram Positive: will remain violet Gram Negative: will remain colorless |
| What are some factors that can affect the accuracy of a gram stain? | -timing of decolorization -too thick -freshness of reagents -improper fixing of smear -changes in organism itself -age of microorganisms/acidity of culture |
| compare simple stains and differential stains | simple-uses just one dye differential-has different reactions with different tissues |
| give 3 clinically important characteristics that are related to the differences in the cell wall of gram positive and gram negative bacteria | -gram + bacteria are generally more susceptible to penicillin and bacteria static dyes -gram + bacteria produces exotoxins -gram + bacteria tend to be more fastidious than gram - bacteria |
Created by:
murra372