Lab II Review Flashcards
- Know the following staining techniques and the stains
used in each: (stains prepared in lab class)
Capsule Stain
- Create a smear. We used S. Liquifacileas Culture using the Nigrosine stain.(it will create the dark background)
- Let air dry
- Heat fix the slide. 2x through the flame gently(reduce overcracking of nigrosine)
- Cover the slide in Crystal Violet Stain for 1 min
- Once min is up turn slide upside down over the sink and rinse with distilled water for 20-25 seconds.
- Blot slide dry with bibulous paper
- Ready to look under the microscope
- Know the following staining techniques and the stains
used in each: (stains prepared in lab class)Endospore Stain-(Schaeffer-Fulton Method)
- Create a smear We used B. Megaterium and a drop of distilled water.
- Let air dry
- Over a Bunsen burner heat a 100ml beaker with water
- Once steam starts place prpared smear slide on beaker. cover the slide with Malachite green stain. Let slide sit over beaker for 5-6 mins. Do not allow the stain to dry. Add more as needed.
- When the 5-6 mins are up. Use clothespin to remove slide and let slide sit for 1 min to cool.
- Once cooled use distilled water to rinse slide to remove the excess malachite green. Book says 30secs but Mrs Zamora says about 2 mins.
- Next cover slide in Safranin for 1 min.
- Rinse the slide with distilled water to remove the excess safranin.
- then blot dry with the bibulous paper
- Ready to look under the microscope
- Know the following staining techniques and the stains
used in each: (stains prepared in lab class)Acid-Fast Stain-(Ziehl-Neelson Method)
- Make a smear using Mycobacterium smegmatis and distilled water
- Let dry
- Gently heat fix the slide 2x only. Let cool.
- Over a Bunsen burner heat a 100ml beaker with water
- Once steam starts place prepared smear slide on beaker. cover the slide with Carbolfuchsin. Let slide sit over beaker for 5 mins. Do not allow the stain to dry. Add more as needed.
- Remove slide with clothespin and cool side for 1-2 mins
- Rinse with distilled water
- Decolorize with acid alcohol. Hold slide at a 45 degree angle add acid alcohol for about 5-10 seconds then rinse with distilled water
- Now stain with Methyl blue for 1 min then rinse with distilled water.
- Then blot dry with bibulous paper.
- Ready to look under the microscope.
- Know the following staining techniques and the stains
used in each: (stains prepared in lab class)Milksmear-Direct Microscope Count
Not on test
- Cell appearance/cell shape using each stain method
Capsule Stain
Bacteria cell stains dark from
Crystal violet (simple stain)
Capsule remains clear
Background stains dark from nigrosine (negative stain)
- Know names of stains.
Nigrosine Crystal violet Carbolfuchsin acid alcohol Methyl blue Malachite green safranin
- Know the importance of Milk Counts as stated by the F.D.A. guidelines.
not on test
- Know the importance of Pasteurization.
not on test
- True Motility versus Brownian Movement.
true motility- darting, spinning/tumbling, wave-like motion
brown in movement- random collision molecules, bacteria cells collide with water molecules, bouncing/ vibrating cells
- Know the following organisms, what test they were used for, correct biological shape, and correct spelling.
Serratia liquifaciens
Bacteria cell stains dark from
Crystal violet (simple stain)
Capsule remains clear
purpose of capsule stain, differential stain used to detect cells capable of producing an extracelluar capsule (usually pathogenic)
- Know the following organisms, what test they were used for, correct biological shape, and correct spelling.
Bacillus megaterium
bacillus shape, to observe the location of an endospore in a sporulating cell.
- Know the following organisms, what test they were used for, correct biological shape, and correct spelling.
Escherichia coli
bacillus shape, to determine if a bacteria is motile and it is very motile,
- Know the following organisms, what test they were used for, correct biological shape, and correct spelling.
Mycobacterium smegmatis
bacillus, to distinguish two groups of bacteria based lipid content in their cell walls: acid-fast and non-acid-fast.
- Importance of Antibiotic Sensitivity Test.
Remember that the purpose of this test us to determine antibiotic treatment for a bacterial infection. These types of sensitivity tests are done typically when a bacteria infection is not responding to the prescribed antibiotic and a doctor needs to determine what antibiotic the pathogen is sensitive to or susceptible to in order to treat the infection.
- Know how to make correct measurements of Clear Zone around
Antibiotic Disc and how to determine Resistance or Susceptibility
to Antibiotic.
Not on test
- Know why the endospore stain and acid-fast stain slides must be heated over a steam bath.
Spores have a thick spore coat and must be steamed to open up spore coat to accept
Stain. Spores stain with malachite green over steam using the endospore stain.
Acid-fast bacteria have a high lipid content in cell wall and must be steamed to open
High lipid content in cell wall to accept stain.
- How many spores are produced per vegetative cell?
1 spore per vegetative cell
- Prepared demo slides viewed in lab:
a. capsule stain
b. simple stain of Bacillus anthracis showing spores
look at ppt slides
- Review the uses of Acid-fast stain and Endospore stain.
harsh methods are needed to stain these organisms, example both had to have a steam bath to open up the spore in endospore stain, and open cell wall in acid-fast stain.
- Know the purpose of each step in the procedure for each stain technique learned in this unit.
1
- Cell appearance/cell shape using each stain method
Endospore Stain-(Schaeffer-Fulton Method)
bacillus, looks like a lollipop
Schaeffer-Fulton Method is designed to isolate endospore by staining endospore gree, and any other bacterial bodies red/pink.
- Cell appearance/cell shape using each stain method
Acid-Fast Stain-(Ziehl-Neelson Method)
Mycobacterium smegmatis bacteria are Acidfast +
and bacillus shape
Acid-fast bacteria stain bright pink from Carbol fuschin stain
