Practical I Flashcards
What are the safety attire required in the lab?
Eyewear, gloves, goggles, aprons, closed-toe and closed-heel shoes, long hair and loose clothing contained.
Eyewear, gloves, goggles, aprons, closed-toe and closed-heel shoes, long hair and loose clothing contained are the requirements of what?
Safety rules for attire in the lab
Disposal rules for the following in the lab:
- Plastic petri plates with bacterial cultures
- Glass tubes with bacterial cultures
- Staining waste
- Broken glass
- Plastic petri plates with bacterial cultures - in biohazard bin
- Glass tubes with bacterial cultures - recycled and autoclaved
- Staining waste - in chemical waste containers
- Broken glass - in sharps container
How do you dispose of plastic petri plates with bacterial cultures in the lab?
In biohazard bin
How do you dispose of glass tubes with bacterial cultures in the lab?
Recycled and autoclaved
How do you dispose of staining waste in the lab?
In chemical waste containers
How do you dispose of broken glass in the lab?
In sharps container
Match the laboratory waste in the first list with the disposal method in the second list.
- Plastic petri plates with bacterial cultures
- Glass tubes with bacterial cultures
- Staining waste
- Broken glass
- Chemical waste containers
- Biohazard bin
- Sharps container
- Autoclaved
- Plastic petri plates with bacterial cultures - biohazard bin
- Glass tubes with bacterial cultures - autoclaved
- Staining waste - chemical waste containers
- Broken glass - sharps container
Why are these characeristics of organisms useful?
They assist in differentiation and identification.
What characteristics of organisms assist in differentiation and identification?
Form, elevation, margin
What is the name and function of #1?
9 is the ocular lenses. They contribute to the total magnification.
What is the name and function of #2
2 is the eyepiece. Connects the ocular lens to the body tube.
What is the name and function of #3?
3 is the body tube. This is the hollow space containing mirrors. Light from the sample travels through the body tube and is reflected through it to the eye pieces.
What is the name and function of #4?
4 is the arm. It connects the body tube to the base and is used to carry the microscope.
What is the name of function of #5?
5 is the rotating nosepiece. It is attached to the other end of the body tube. The nosepiece can rotate to allow the use of four different objective lenses.
What is the function of #6?
6 are the objective lenses. There are four.
- Scanning lens: 4X. Always start with this lens.
- Lower power lens: 10X
- High power lens: 40X
- Oil immersion lens: 100X. Always use oil to prevent scattered light from causing a blurry image.
What is the function of #7?
7 is the mechanical stage. This supports the slide.
What is #8 and what function does it serve?
8 is the condenser. It bends the light to focus on the specimen.
The part below it, #9 is the iris diaphram. Remember, the condenser is above the iris diaphram which serves as the aperature.
What is the name and function of #9?
9 is the iris diaphragm. It acts like an aperature that can be opened or closed to adust the light passing from the leamp to the sample.
It is below the condenser that bends the light to focus on the specimen.
What is the name and function of #10?
10 is the lamp. The lamp provides illumination and is controlled by the rheostat.
What is the name and function of #11?
11 is the base. This is the support of the microscope.
What is the name and function of #12?
12 is the rheostat. It controls the amount of light emitted by the lamp.
What is the name and function of #13?
13 is find focus. It raises and lowers the stage in small increments.
What is the name and function of #14?
14 is the course focus knob. This raises and lowers the stage in bigger increments. Only used with the 4X objective.
Why is it important to recenter the specimen when changing objectives?
The field of vision is smaller with larger objectives and the field of vision will be lost if the specimen isn’t centered.
When focusing with an oil objective, what are the steps?
- Start with 4X objective
- focus
- adjust light
- center
- 10X objective
- refocus
- adjust light
- center
- Change to 40X
- refocus
- adjust light
- center
- add oil to the specimen
- refocus
Name organism and relate to group:
- protists
- algae
- helminths
- fungi
Amoeba; Protist
Name organism and group (kingdom)
Rhizopus; fungi
Name organism and group (kingdom)
Spirogyra; algae and protists
Name organism and group (domain)
Anabeana; bacteria
Name organism and group
Taenia pisiformis; helminths
What domain does each belong to?
- Amoeba
- Anabeana
- Rhizopus
- Spirogyra
- Taenia pisiformis
- Amoeba - Eukaryote
- Anabeana - Bacteria
- Rhizopus - Eukaryote
- Spirogyra - Eukaryote
- Taenia pisiformis - Eukaryote
Calculate size of cell using calibration provided under 40X objective.
1 optical unit (O.U.) x 40X calibration of 2.5 μm = 2.5 μm
(do not consider the 10X ocular lens)
Calculate size of cell using calibration provided under 4X objective.
1 O.U. x 25 μm = 25 μm
Find the size of the cell with 100X objective.
1 O.U. x 1 μm = 1 μm
Find the size of the cell with 10X objective.
1 O.U. x 10 μm = 10 μm
What is broth and how is it inoculated?
Broth is a medium that contains nutrients suspended in a liquid solution; inoculated with a loop.
What is the medium that contains nutrients suspended in a liquid solution and is inoculated with a loop?
Broth
What is agar and how would you inoculate it?
Agar is the solidifying agent in a medium that contains nutrients found in broth; inoculate with a loop or a spreader.
How do you calculate the magnification of a specimen with a microscope?
Ocular lens power X objective lens power
Ocular lens is usually 10X.
- With 4X (scanning lens): 10 X 4
- With 10X (low power lens): 10 X 10
- With 40X (high power lens): 10 X 40
- With 100X (oil immersion): 10 X 100
Identify each tube as broth, agar slant or agar deep.
Broth, agar slant, agar slant, agar deep
What is this called?
Agar plate
How would you inoculate each?
Loop, loop, loop, deep
With what do you inoculate an agar plate?
Loop or streak plate method
How many streaks are on this agar plate?
Three
Why is the streak plate technique used?
To isolate single colonies from single cells. Because of this, a single colony contains only one type of cell.
What is the process by which to complete a streak plate culture?
Know procedure:
- Flame loop
- Dip in original culture
- Spread from sector 1 to 2
- Flame loop
- Spread from 2 to 3
- Flame loop
- Etc.
Only dip into original culture once in beginning.
How many times do you dip the inoculating loop into the culture when completing the streak plate technique?
Once in the beginning.
What is the goal of the streak plate technique?
To obtain single colonies
What are the characteristics of negative stain?
- Negative staining is simple stain
- Won’t reveal anything about cell walls
- Stains background
- Stain is large, uncharged molecules
- Example: India ink
Which type of staining method does this and what type of stain is it?
- Won’t reveal anything about cell walls
- Stains background
Negative staining; simple stain
What will a negative stain show you?
Size, shape and arrangements of organisms.
Is negative staining simple or differential?
Simple. Does not show cell walls.
What is the stain in this image?
Simple stain with a large, uncharged stain (such as India ink)
Is this simple or differential staining?
- Simple stain; does not differentiate
- Stain with a single stain (such as crystal violet, safranin, methylene blue)
What is a stain with a single stain (such as crystal violet, safranin, methylene blue)
Simple stain
What does a simple stain reveal about a cell?
See cell size and shape but nothing else like cell wall.
What are three simple stains?
- Crystal violet - stains purple, primary stain in Gram stain. In Gram stain, Gram+ cells will remain purple from crystal violet while crystal violet will wash out of the wimpy peptidoglycan in the gram- cells upon washing with alcohol.
- Safranin - stains pink/red, counterstain in Gram stain. In Gram staining, gram- cells will take on the safranin after washing with alchohol.
- Methylene blue - stains blue, counterstain in acid-fast staining. In acid-fast staining, cells with a high concentration of mycolic acid in their peptidoglycan layers will remain pink from carbolfuchsin. Negative cells will turn blue with counterstain.
Remember, positive cells retain color of primary stain, not counterstain, in Gram and acid-fast staining.
What is simple staining?
To stain with a single stain such as crystal violet, safranin, or methylene blue.
To stain with a single stain such as crystal violet, safranin, or methylene blue is called what?
Simple stain
What is an example of a stain used in negative staining?
India ink
Size, shape and arrangement are indicative of what type of staining method?
Simple staining.
Simple staining reveals what about an organism?
Size, shape, arrangement.
Is capsule stain a simple or differential staining method?
Differential. It uses a negative stain (stains background) and a simple stain (stains cells).
What does a capsule stain reveal about the cells?
Whether cells have an outercalyx or capsule. The capsule will not stain from either the negative stain or the simple stain and will appear as a halo. No halo, no capsule.
What staining method reveals a capsule around cells?
Capsule staining.
What makes the capsule around a cell appear as a halo?
The capsule is a type of glycocalyx that does not stain.
When performing a capsule stain, what will appear around cells with a glycocalyx?
A halo. Cells without a capsule will have no halo.
What is the procedure for Gram stain?
- Stain with crystal violet
- Treat with iodine
- Decolorize with ethanol
- Counterstain with safranin
Result: Positive cells are dark purple (from crystal violet), negative cells are pink (from safranin)
What is the procedure for acid-fast staining?
- Stain with carbolfuchsin and heat
- Decolorize with acid-alcohol
- Counterstain with methylene blue
Result: Positive cells are pink (from carbolfuchsin), negative cells are blue (from methylene blue).
What about a cell makes it stain acid-fast positive?
High concentration of mycolic acid in the cell wall.
What are the characteristics of Gram-positive cells with regard to:
- Peptidoglycan layers in their cell wall
- Membrane
- Teichoic acid and lipoteichoic acid
- End result of Gram staining
- Gram + cells have thick, peptidoglycan layer in their cell wall
- Have no membrane
- Have teichoic acid and lipoteichoic acid
- Stain purple (with primary stain/crystal violet) with Gram stain
What are the characteristics of Gram-negative cells in regard to:
- Peptidoglycan layers
- Molecules in outer membrane
- Plasma and outer membrane
- Proteins in outer membrane
- Gram stain color result
- Have one if any peptidoglycan layer
- Have lipopolysaccharide in outer membrane
- Have periplasmic space between plasma and outer membrane
- Have porin proteins
- Stain pink (from safranin/counterstain) with Gram stain
What type of staining technique are Gram and acid-fasting staining?
Differential staining methods
In what staining technique is safranin used and where is it used?
Used in Gram stain.
Is counterstain.
In what staining technique is methylene blue used and where is it used in the technique?
Acid-fast staining.
Counterstain.
In what staining technique is crystal violet used and where is it used in the technique?
Gram stain.
Primary stain.
In what staining technique is carbolfuchsin used and where is it used in the technique?
Acid-fast stain.
Primary stain.
What are the results in acid-fast staining?
Positive are pink.
Negative are blue.
What are the results in Gram staining?
Positive are purple.
Negative are pink.
Name the four flagella arrangements in the image from top to bottom.
What is the name of a cell flagella arrangement with a tuft of flagella?
Lophotrichous
What is the name of the flagella arrangement where a single flagella is located at each pole?
Amphitrichous
What is the flagella arrangement with flagella all over the exterior of a cell?
Peritrichous
What is the name for the flagella arrangement of a cell with one flagella at one pole?
Monotrichous
In this specimen, what type of flagellar arrangement is this?
Peritrichous
In what type of media are bacterial grown to view motility and how are they inoculted?
In deeps (motility media) with an inoculating needle
What will motility media called a deep that is inoculated with a needle intend to show?
If the organisms can swim away (have flagella).
(Flagella are present in the tube on the right - includes axial flagella.)
In which deep would you expect to find organisms without flagella?
On the left.
In what deep would you expect to find organisms with flagella, even axial flagella?
On the right.
What are the two methods used to culture anaerobes?
FTM (fluid thioglycollate medium)
Bio-Bag (container lacking oxygen)
What are cultures grown in this media called, left to right?
Obligate aerobe, obigate anaerobe, facultative anaerobe
Mark the area of growth in each tube and the type of microbes.
Obligate aerobe, obligate anaerobe, facultative anaerobe
What media is this test grown in?
Cultures are grown in fluid thioglycolate media (FTM)
What can you tell about the organisms growing in this media?
They are obligate anaerobes. They do not grow in oxygen because they have no enzymes that protect them from oxygen.
What can you tell about the organisms growing in this media?
They are facultative anaerobes. They can grown in no oxygen or high oxygen because they are anaerobes that have enzymes that protect them from the effects of oxygen in the atmosphere.
What can you tell about the organisms growing in this media?
They are obligate aerobes. They can only grow in the presence of oxygen.
Where in these tubes is the least amount of oxygen and what two types of organisms can grow in this environment?
The bottom.
Obligate anaerobes and facultative anaerobes.
Where in these tubes is the most amount of oxygen and what type of organisms grow there?
At the top.
Obigate aerobes and facultative anaerobes grown in oxygen.
Why are factultative anaerobes able to grow in high oxygen environments?
Because they have enzymes that protect them in high oxygen environments.
Why is fluid thioglycolate media (FTM) used to culture anaerobes?
Because it reduces oxygen in media.
Where do organisms that can tolerate toxic oxygen grow in a Bio-Bag?
Outside the bag.
These type of organisms grow outside the Bio-Bag.
Those that can tolerate toxic oxygen.
Where do organisms that can tolerate toxic oxygen grow in a Bio-Bag?
Outside the bag.
Where do organisms that can tolerate toxic oxygen grow in FTM?
At the top of the tube.
At the top of the tube of FTM is where what type of organisms grow?
Those that can tolerate toxic oxygen.
Where do organisms that can make ATP without oxygen grow in FTM?
At the bottom of the tube.
At the bottom of the tube is where what organisms grow in FTM?
Those that can make ATP without oxygen.
Where do organisms that can make ATP without oxygen grow with a Bio-Bag?
Grows inside the Bio-Bag
What organisms grow inside a Bio-Bag?
Those that can make ATP without oxygen.
What is the aliquot in the dilution formula?
The cells (stuff) being diluted.
What is the denominator of the dilution factor?
Aliquot + volume in tube.
What is the dilution factor for this?
Dilution factor is 1/10
What is the dilution factor?
1/10
What is the importance of heat fixing a slide?
Heat fixation adheres the cells to the slide and coagulates the bacterial proteins, effectively killing the bacteria.
What technique results in this: Adheres cells to the slide and coagulates the bacterial proteins, effectively killing the bacteria.
Heat fixing a slide
Give the characteristics of E. coli.
Escherichia coli are
- Gram negative (stain pink)
- Acid-fast negative (stain blue)
- Rod-shaped (bacillus)
- Single cell arrangement
Which organism has these characteristics:
- Gram negative (stain pink)
- Acid-fast negative (stain blue)
- Rod-shaped (bacillus)
- Single cell arrangement
Escherichia coli
What characteristics do Staphylococcus aureus have?
Staphylococcus aureus are:
- Gram positive (stain purple)
- Acid-fast negative (stain blue)
- Cocci shaped (cocci)
- Grapelike clusters (staphylo)
What organism has these characteristics?
- Gram positive (stain purple)
- Acid-fast negative (stain blue)
- Cocci shaped (cocci)
- Grapelike clusters (staphylo)
Staphylococcus aureus
Which tube contains which of the organisms below and what type of aerobe are they?
- E. coli
- C. sporogenes
- P. aeruginosa
Left to right:
- P. aeruginosa* (obigate aerobe)
- C. sporogenes* (obligate anaerobe)
- E. coli* (facultative anaerobe)
What is the name of a single coccus cell, two coccus cells and a chain of them?
Coccus, diplococci, streptococci
What is a grapelike cluster of round cells called?
Staphalococcus
What are the possible arrangements of rod-shaped bacteria and what is the shape called?
Shape: bacillus
One: Single bacillus
Two: Diplobacilli
Chain: Streptobacilli
Other organisms observed:
- Bacillus subtilis*
- Staphylococcus epidermidis*
- Spirilum volutans*
- Bacillus subtilis* - rod-shaped, clusters
- Staphylococcus epidermidis* - coccus, grapelike clusters
- Spirilum volutans* - spirals
