Micro Lab Exam 1 Flashcards
How much media for a small test tube?
5 ml of media
How much media for a large test tube?
10 ml of media
How much media for a petri plate?
25 ml of media
A petri plate is a two step process. What are they?
- Media is placed in large test tube with the cap on so it can be sterilized.
- Once sterilized it can be stored in the test tube until it is to be transferred to the petri plate.
Negative Stain - what is the result?
cell remains clear and background is dark
What type of stain is Nigrosine?
Negative stain
What charge is Nigrosine stain?
- (negative) Charge
What is the % of the following bacteria cells in the human mouth? Coccus, Bacillus & Spirochete
Coccus 100%
Bacillus 80%-90%
Spirochete 10%-20%
What is the importance of the quadrant streak plate inoculation?
- Observe bacteria colonies
2. separate a mixed culture of bacteria into separate colonies
What is the importance of heat fixing a smear before staining?
- Kill bacteria
- Adhere cells to slide
- Preparing cells for stain
What was the stain we used for the simple stain?
Methylene blue
What are the 4 simple stains listed in our lab book?
Crystal violet
Methylene blue
Safranin (red)
carbolfuchsin (red)
What is the process to do a simple stain with methylene blue?
- Mix the bacteria with distilled water on clean slide. Smear all over slide. Set aside and let dry.
- Place slide over sink/tray and use a couple drops of methylene blue. Let stain sit for 1 min on slide.
- Rinse slide with distilled water to remove excess stain.
- Blot excess water from slide and let dry. Can use paper towel or Bibulous paper.
What is the process to do a negative stain with Nigrosen?
- Use a clean slide and place a drop of Nigrosen on one end of the slide.
- Use a sterilized inoculating loop(we used toothpicks) and mix the bacteria with the Nigrosen for about 15 seconds.
- Re sterilize the loop in a flame before setting down.(throw out toothpick)
- Using a clean slide. touch the short end on the slide with the stain and a 45 degree angle and drag it toward the drop of stain/bacteria and create the smear.
- Remove and rise the second slide and allow the smeared slide with bacteria and Nigrosen air dry completely.
- Then view under the microscope.
What stain did we use for our negative stain?
Nigrosen
What stains are used for the gram stain?
Crystal Violet
Gram’s Iodine
95% ethanol (Alcohol)
Safranin
What is the process to do a gram stain?
- Need a slide with a fixed smear of bacteria.
- Over a sink or tray cover the smear with Crystal Violet for 1 minute
- When minute is up rinse with distilled water.
- Then cover the smear with the Gram’s Iodine for 1 minute.
- When minute is up rinse with distilled water.
- Decolorize with the 95% ethanol (alcohol). Hold slide at a 45 degree angle and count to 10 then immediately rinse with distilled water.
- Last stain is the Safranin. Cover slide and wait for 1 minute.
- When minute is up rinse slide with distilled water.
- Finally blot slide gently with paper towels or bibulous paper.
- Then examine under microscope using oil immersion lens
The process of heat fixing a smear before staining.
- Place small amount of distilled water on slide and then smear bacteria in water and cover the slide. Attach a cloth spin to the end of the slide to prevent burning of our fingers.
- Using a Bunsen burner place the flame high. Quickly move slide through the middle of the flame bacteria side up. test the heat of the slide on our hand/wrist. Do this a total of 3 times.
- Let cool and it is ready to stain.
In doing a gram stain what is the purpose of the Crystal violet and what color will it be for G+ and G-?
The purpose is to stain the bacteria cells purple
G+ / Purple G- / Purple
In doing a gram stain what is the purpose of the Gram’s Iodine and what color will it be for G+ and G-?
The purpose is it binds to the Crystal Violet.
G+ / Purple G- / Purple
In doing a gram stain what is the purpose of the Alcohol and what color will it be for G+ and G-?
The purpose is to dissolve the LPS membrane
G+ / Purple G- / Clear
In doing a gram stain what is the purpose of the Safranin and what color will it be for G+ and G-?
The purpose is it is a counter stain. It will stain G- cells.
G+ / Purple G- / Pink red
