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How does the refractive index of immersion oil compare with that of glass?
Immersion oil has the same refractive index compared with that of glass. Having the same refractive index as glass prevents light loss due to diffraction. Oil immersion should be used between the slide and 100x objective lenses this is a special oil that has the SAME refractive index as glass. When placed between a specimen and objective lens, the oil forms a continuous lens system that limits the loss of light due to refraction
How do you calculate the total magnification of a specimen seen through a compound light microscope?
The total magnification of a compound microscope is determined by multiplying the power of the ocular lens times the power of the objective lens used. Therefore the magnification of a microscope in which oil immersion lens is being used is : 10 X 10= 100
What does the term "parfocal" mean?
"Parfocal" means that the image will remain in focus when changing from a lower-power objective lens to a higher-power lens. * only minimal focusing should be necessary with the fine focus adjustment
Why is it advisable to start with the low power lens first when viewing a slide?
To enable you to explore the slide to look for the object that you are planning to study
In the lab we looked at either prepared slides or fresh wet mounts of the following organisms: Paramecium, Euglena, Spirogyra, Oscillatoria, Amoeba, Stentor. Classify them into the appropriate classification group; protozoans, algae, or cyanobacteria.
Paramecium, Amoeba, Stentor= Protozoans
Euglena, Spirogyra= Algae
Compare and contrast the three mechanisms of motility displayed by protozoans.
Flagella, Cilia, Amoeba
What is flagella?
Long whip-like structure that have a 9 +2 arrangement of microtubules. During asexual reproduction most flagellated cells divide longitudinally
What is Cilia?
short hair-like projection propel the cell forward and backwards. Some cells attach to a substrate and use cilia to create feeding currents around the mouth
What is Amoeba?
most move by formation and extension of transitory pseudopodia, which are cytoplasmic extensions. Amoebas are predators and are usually feed on bacteria and protist by engulfing them forms a food vacuole around the prey
Give three reasons why it is important to practice aseptic technique in the microbiology lab
-Insure that no contaminating organisms are introduced into culture material when the latter are inoculated or handled in some manner
-Insures that organisms that are being handled do not contaminate the handler or others who may be present
-Its use means that no contamination remains after you have worked with the cultures
Why should agar Petri plates always be inverted during incubation?
Prevents moisture from condensing on the agar surface and spreading the inoculated organisms
Why is the Bunsen burner used during inoculation of a tube culture?
it is used to destroy any organisms that remain on the implements of the test tube to reduce contamination
Describe how you would go about preparing a streak plate.
-Disinfect your table
-Label the bottom surface of a sterile petri plate with your name and date
-Liquefy a tube of nutrient agar, cool to 50 degrees C, and pour the medium into the bottom of the plate
-Streak the plate by the method that was shown on lab day
-Shake the culture tube from side to side to suspend organism.
-Heat wire to red-hot. Flame the hand slight also
-Remove the cap and flame the neck of the tube
-After allowing the loop to cool, remove loopful or organisms. Avoid touching the side of the tube
-Flame the mouth of the culture tube again
-Return the cap to the rube and place the tube in the test tube rack
How would you determine if your agar slants contain pure cultures?
If the organism contain only a single kind of organism whereas mixed cultures contains more than one kind of organism
How would you confirm their purities?
Isolated colonies can then be subcultured and stains prepared to check for purity
Why is dilution such an important practice in the microbiology lab?
It is important practice in the microbiology lab because both procedures involve diluting of the bacterial cells in a sample to an end point where a single cell divides giving ride to a single pure colony, which gives you an identical progeny of the original cell and can be picked and used for further study of the bacterium
What is the purpose of heat fixation when preparing a smear?
So that the cells adhere to the slide and are not washed off during the different staining and washing procedures
List the basic steps in the preparation of a smear from a liquid media.
-Two loopfuls of liquid contain organisms are placed in the circle that you drew at the bottom of the slide
-Organisms are dispersed over entire area of the circle
-The smear is allowed to dry at room temperature
-Slide is passed through flame several times to heat-hill and fix organism to slide
List the basic steps in the preparation of a smear from a solid media.
-Two loopfuls of water are placed in center
-A very small amount of organism is dispersed with inoculating loop in water over entire area of circle
-Smear is allowed to dry
-Slide is passed through flame several times
What is the difference between a simple stain and a differential stain?
A simple stain is : the use of a single stain to color bacterial cells
A differential stain: these staining reactions take advantage of the fact that cells or structures within cells display dissimilar staining reaction that can be distinguished by different dies
Why did we practice putting three smears on one microscope slide- on bacterial type on the left, another type on the right, and a mixture of the two in the middle?
to see which bacteria took which stain in order to find out which stain came from which bacteria, for example which gram positive or which gram negative, it gives you the ability to separate and distinguish which bacteria is which
Summarize the steps in the Gram staining procedure, including the reagents used and the length of time each is used.
-Cover the smear with CRYSTAL VIOLET and let stand for 1 minute.
-Briefly wash off stain, using a wash bottle or distilled water. Drain off excess water [2 seconds0
-Cover the smear with the Gram's Iodine solution and let it stand for 1 minute
-Wash off the Gram's Iodine, decolorize with alcohol for 2 to 5 seconds
-Stop Decolorization by washing the slide with a gentle stream of water
-Cover the smear with safranin for one minute
- Wash gently for a few seconds, blot dry with bibulous paper, and air dry
-Examine the slide under oil immersion
What is the result of the gram stain?
Gram Positive Stain: Purple cells
Gram Negative Stain: Pink cells
What are the uses of a gram stain?
Divides bacteria into one of two groups:
Gram-positive or Gram-negative
What is the result of the Ziehl-Neelsen Acid-Fast Stain?
Non-Acid Fast: Blue Cells
Acid-Fast: Red cells
What are the uses of the Ziehl-Neelson Acid-Fast stain?
Distinguishes members of the genera. Mycobacterium and Nocardia from other bacteria; Divides bacteria into one of two groups; Acid Fast and/or Non-acid fast
What are the uses of the Schaeffer-Fulton Spore stain?
Allows visualization of hard-to stain bacterial Endospores such as members of genera Clostridium and Bacillus
What is the result of a capsular stain?
clear halo-like area surround pink cells
no clear area surround pink cells
What are the uses of the capsular stain?
Demonstrates the presence of capsule formers ** the capsule itself does not stain
Describe the difference in the appearance of a motile versus a non-motile bacterial culture after incubation in a semisolid agar medium? Drawings are helpful!
-If the organisms are motile, they will swim away from the line of inoculation into the uninoculated surrounding medium, causing the medium to be turbid
-Non-motile bacteria wil be found only along the line of inoculation
What is the major organelle of motility in bacteria? Give an example of a bacterium that has the structure.
The major organelle of motility in bacteria is flagella, and example of a bacterium that has this structure is Proteus Vulgaris
In the lab we used fluid thioglycollate medium (FTM). Why is this special medium idea for this lab?
because it supports the growth of both aerobic and anaerobic bacteria. It also has an indicator of the the presence of oxygen the dye then becomes pink
Compare the positions in the FTM tube of an obligate anaerobe, facultative anaerobe, and an obligate anaerobe with their different oxygen needs.
-Obligate aerobe- at the top of the surface, free oxygen for aerobic respiration
-Facultative anaerobe- scattered throughout the tube; carry on aerobic metabolism when oxygen is present but shift to anaerobic metabolism when oxygen is absent
-Obligate anaerobe- at the bottom of the tube; killed by oxygen
What method did we use in the lab to determine bacterial numbers in sample?
The standard plate or viable count method
What does the standard plate or viable count method involve?
This involves a sample being diluted in a series of dilution blanks, they are then plated onto media and the numbers of colonies counted after incubation for 24-48 hours. It is assumes that the bacterial cels are diluted to an end point where a single cell divides giving rise to a visible colony on a plate
What is the acceptable range of colony forming units?
30 to 300 colonies are considered statistically valid
What are the problems when the CFU's are above this range?
If the CFU is greater than 300 than there is a probability that overcrowding on the plate could have inhibited some cells from growing
What are the problems when the CFU's are below this range?
If less than 30 CFUs could involve sampling error and an underestimate of numbers
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