Lab Unit 1 Flashcards
What type of footwear is allowed in lab?
Closed toed shoes only
Where must equipment go after you are finished with your lab assignment?
In the red tote box
What must you do to the counter tops before and after lab?
Wipe them down with disinfectant.
Where must you dispose of any hazardous materials including toothpicks, Petri dishes, gloves, etc?
The biohazard bag. Everything else goes into regular trash, including paper towels used to dry your hands.
When putting away or retrieving glassware such as flasks, beakers, test tubes what must we first do?
Remove all grease marks.
If there is a spill what are the steps we take to properly clean them?
Cover them with paper towels, saturate the spill with disinfectant for 10 mins.
Where must we dispose what we used to clean it?
In the biohazard bag.
What must be done before using your microscope?
Ensure that is has been put away correctly.
What must be done after using your microscope?
Ensure that is has been put away correctly.
Are you allowed to have food, drink, water bottles, or gum in lab?
No.
Where is the first aid kit located?
In the cabinet by Andrea’s door.
What are the four techniques of inoculation?
Streak plate
Streak for Isolation
Spread plate
Pour plate
These two techniques are quantitative methods used to determine the number of bacteria in a sample.
Spread plate
Pour plate
What is the spread plate technique?
A small amount (several drops) of a previously diluted sample is spread over the surface of a solid medium (Petri dish) using a spreading rod, which is a sterile glass rod bent at 90°.
What is the pour plate technique?
A small amount of diluted sample is mixed with melted agar and poured into empty, sterile Petri dishes. A serial dilution of the bacterial sample is performed first, then a small amount of each dilution is pipetted into the empty Petri dishes, then the melted agar is added. This allows bacteria to be inoculated throughout the media.
In this method the inoculated loop or swab is used to streak the sample many times in a zig-zag over the surface of a solid culture medium in a Petri dish.
Streak plate method
This technique is used to take a mixture of bacteria and separate them out into pure cultures to identify each organism.
Streak for isolation
When using streak for isolation your petri dish should be divided into how many parts?
4 quadrants
During a streak for isolation, after you finish EACH streak which way must you turn your plate?
Counter-clockwise
Why is this technique so commonly used by doctors?
The physician needs to know what EXACTLY the organisms are so he/she may treat the patient accordingly.
What does this method result in when the sample are taken?
Fewer organisms are dragged into each quadrant, so that the last quadrant does not overlap its colonies of organisms.
What happens if the streak is successful?
In a few days you will see separate colonies in the last quadrant.
What happens if it was unsuccessful?
Different looking colonies will be overlapping each other in the last quadrant.
If your streak was unsuccessful, what could have been done incorrectly?
Too many organisms in the first quadrant and they too got dragged along.
Plates must be incubated upside down, why?
It prevents condensation from developing on the inside lid, and then dropping onto your agar, mixing the organisms.
It also ensures you are able to read its labels.
When do you use an inoculation needle?
If you need to take a pure colony from a plate. You need a needle.
What 3 things to look for in nutrient slant characteristics?
- Is there turbidity (cloudiness)?
- Is there growth only at the top (pellicle), only at the bottom (sediment), flakes in the middle (flocculent), or growth throughout (uniform turbidity)?
- What color is it?
What are the 3 different types of plate characteristics?
- Margins (look at the surface of the plate)
- Texture (look at the surface of the plate)
- Pigmentation (color of plate)
What are 6 types of colony margins?
- Filiform (thread-like edge)
- Arborescent (tree-like branches)
- Beaded (starts smooth, breaks into individual colonies at top)
- Effuse (edges are not clearly defined)
- Rhizoid (root-like branches)
- Echinulate (serrated edge)
What are the 4 types of colony texture?
- Flay, dry (Bacillus megatarium)
- Spreading (proteus, pseudomonas)
- Crusty, waxy (mycobacterium)
- Transparent
What to look for in pigmentation?
- Yellow
- White
- Green
- Etc.
What are the 3 different colony configuration types?
- Round
- Irregular
- Rhizoid, arborized
What are the 4 different colony elevation types?
- Convex
- Flat
- Raised
- Umbonate (belly button protrusion)
To what are nutrients added to make media for bacteria cultures?
Agar (seaweed product)
What type of nutrient medium does not solidify?
A broth
What is it called when you intentionally introducing microbes onto nutrient agar and into nutrient broth?
Inoculation, which means having a tiny amount of a bacterial culture streaked across the surface.
What happens after inoculation?
The plate or tube is usually incubated for at least 24 hours to encourage growth of the sample.
What two ways are culture media classified?
Consistency and contents
What are 4 media classifications based on their contents?
- All-purpose
- Selective
- Enriched
- Differential
What does consistency refers to?
A liquid, solid, or semi-solid.
What 2 things does semi-solid allows us to do?
To see if the microbe is motile (can move), and to see if the microbe is aerobic or anaerobic.
What organisms are not supported by nutrient agar (NA)?
Fastidious (hard to grow) organisms.
What are the 3 types of isolation media?
- Selective media
- Enrichment media
- Differential media
What is selective media?
Selects for a particular type of organism to grow.
What do selective media contain?
What does it do?
It contains chemical that prevent the growth of unwanted bacteria without inhibiting the growth of the desired organism.
What is an example of selective media?
Sabouraud’s Dextrose Agar (SDA)
What does SDA contain?
It has a high sugar content and an acidic pH.
What is the classification of SDA media?
Selective, or Isolation media
Why is that?
It isolates molds and yeasts, which do well in high sugar content, whereas bacteria inhibited.
What natural substance is a good a bacterial preservative?
Sugar
Do jams, jellies, and preserves grow bacteria?
No
Are molds aerobic or anaerobic?
Aerobic
How do molds get onto our food?
Microscopic air-borne spores.
Should you throw out all of the food when you suspect mold? Why?
Yes. Some species produce toxins that cause liver cancer.
What pH do molds and yeasts prefer?
Acidic
What media was originally designed to isolate dermatophytes?
SDA (Sabouraud’s Dextrose Media)
What is a fungus that can infect skin, nails, hair, and are opportunistic pathogens?
Dermatophytes
What produces ringworm, especially athlete’s foot?
Dermatophytes
What microbes are known for causing infections in women, diabetic, and cancer patients?
Yeasts
Why are diabetics more prone to infections?
They are immunocompromised due to their high glucose levels.
How do you isolate a fungus from the skin?
Scrape off a little skin and place in SDA
What type of media contains various nutrients that allow the investigator to distinguish on bacterium from another by how they metabolize or change the media with a waste product?
Differential media
What media will contain a substance that turns yellow if one group of organisms are present, or red if another group of organisms are present?
Differential media
When using differential media, how can you tell which group of bacteria there are?
By the color the plate turns
What type of media has added nutrients such as vitamins and amino acids to enhance the growth of desired bacteria?
Enriched media
What type of media is used to grow bacteria which are hard to grow, known as fastidious organisms?
Enriched media
Are most pathogenic organisms fastidious?
Yes
What is an example of enriched media?
Blood agar
What media is the same as NA, but has no agar?
Broth
What are made exactly as NA except poured into tubes instead of plates?
Slants
What is the benefit to using a slant?
We can inoculate just the top of the slant to get growth of aerobic bacteria, or we can stab a needle of bacteria into the tube to see if there are any microbes that can grow without air (anaerobic).
What is the benefit to using tubes over Petri dishes?
A Petri dish will only keep fresh for 3 weeks, then they dry out. Tubes last for a long time in the refrigerator, and are useful for making stock cultures.
How to use a microscope:
Arm should face you as you lift it up. Hold one hand under the base, and turn the arm away from you. Fold up dust cover and put it in drawer.
The basic frame of the microscope consists of what 4 parts?
Base, stage, arm, and body tube
What raises and lowers the condenser?
The substage adjustment knob
When you are done with the microscope, leave the condenser in the highest position. This is called…
Racking up the condenser
What is the eye piece called?
Ocular
What magnifies ten times and focuses the image on the retina?
Ocular
How do you clean the oculars?
Squirt alcohol onto lens paper and wipe, then dry with lens paper.
Which ocular has a diopter adjustment ring?
The left ocular
What is the purpose of the diopter adjustment ring?
To allow the left eye to be focused independently of the right eye.
What revolving piece holds the objectives?
The nosepiece
What is the smallest objective lens?
The scanning (red ring) objective
What is the magnification of the scanning objective?
4x
How do you find total magnification?
Multiply the ocular magnification by the magnification of the objective.
What is the total magnification of the scanning objective?
40x
What is the total magnification of the low power objective?
100x
What is the total magnification of the high dry objective?
400x
What is the total magnification of the oil immersion objective?
1000x
What is the total magnification of a microscope that has a 10x ocular, when using the scanning lens?
Ocular x lens = Total magnification
10 x 4 = 40x
What is the total magnification of a microscope that has a 10x ocular, when using the low power lens?
10 x 10 = 100x
What is the total magnification of a microscope that has a 10x ocular, when using the high dry lens?
10 x 40 = 400x
What is the total magnification of a microscope that has a 10x ocular, when using the oil immersion lens?
10 x 100 = 1000x
What is the working distance?
The distance between the stage and the objective lens.
What does parfocal mean?
Once you are focused with the scanning objective, you are focused with all of the objectives.
What takes light from the lamp and makes the rays into a point on the slide?
The condenser
What opens and closes like the iris in your eye (pupil)?
The iris diaphragm lever
Why does the iris diaphragm lever open and close?
To regulate the amount of light allowed in.
When changing from low to high power, what needs to be done to the iris diaphragm?
It needs to be opened.
What is required for increasing magnification?
MORE LIGHT
What are the four things the iris does?
Brightness
Contrast
Depth of field
Resolution
When the iris is open, what happens to contrast?
Decreases
When the iris is open, what is in focus?
Only the foreground
When the iris is closed, what happens to the depth of field and what is in focus?
Depth of field increases.
Everything is in focus.
What is the working distance?
The length between the tip of the objective lens and the stage
Which objective lens do you start with?
Lowest power
As you increase magnification, what happens to working distance?
Decreases
Why does this trend occur?
Because the objectives are taller as you go up in magnification.
Which objective has the largest field of view?
the scanning objective
Which objective has the greatest working distance?
the scanning objective
Which objective has the smallest field of view?
oil immersion lens
Which objective has the shortest working distance?
oil immersion lens
What is the substage adjustment knob used for?
Moves condenser up and down (when down, there is poor resolution).
What two things affect the resolving power of a microscope?
The numerical aperture of the lenses.
The wavelength of light.
What is the diopter ring used for?
Allows left eye to focus independently.
What organisms are so small, so they always need 1000x and oil to be seen?
Bacteria
What are three basic shapes of bacteria?
Spiral
Cocci (singular is coccus)
Bacilli (singular is bacillus; also known as rods)
How do you clean up oil from the microscope?
Use alcohol and Kim wipes to remove oil from the microscope stage and the slide before you return it to the slide tray. Use alcohol and lens paper to remove the oil from the objective.
True or false: When putting away the microscope, the AC (power) cord is wrapped loosely behind the scope.
True
True or false: When putting away the microscope, the arm should face away from your body and the dust cover should be in place.
False; the arm should face your body.
True or false: When putting away the microscope, the condenser should be racked up (placed in its highest position).
True
True or false: When putting away the microscope, the toggle switch for power should be off (it is located in the front or side).
True
True or false: When putting away the microscope, the voltage should be turned to the highest setting.
False; the voltage should be turned to zero or the lower setting.
True or false: When putting away the microscope, the stage should be racked down.
True
True or false: When putting away the microscope, the scanning objective 4x (red ring lens) should be out of place.
False; the scanning objective 4x (red ring lens) should be in place.
When putting away the microscope, what needs to be cleaned from the stage and the microscope in general?
Oil and fingerprints
When putting away the microscope, what needs to be used to wipe the ocular lens?
Lens paper only
When putting away the microscope, where should the eyepieces face?
The blue side of the scope
When putting away the microscope, what should be done to the screw?
Tighten it a bit.
When putting away the microscope, how can you tell that the objectives are clean?
If they are not oily on the outside or blurry to look through.
What microbes are classified as potentially pathogenic (cause disease) if a person is inoculated with a large dose or if the person is immuno-compromised?
BS2 (biosafety level 2) microbes
How are these microbes killed?
Autoclaving (steam heat sterilization)
How should these microbes be handled?
By aseptic technique and with safety equipment under the hood.
What gear do these microbes require?
A space suit type of gear.
What type of air pressure does a room dealing with these microbes require?
Negative pressure
Why does the room need to have this type of air pressure?
To prevent airborne pathogens from escaping through the door. They are sucked up the hood.
How often is the air in this room need to be circulated?
About every eight minutes
How can the bacterial inoculate be transferred?
Using an inoculation loop (a wire with a small loop at one end and a handle at the other) or an inoculation needle (wire on a handle, but without a loop on the end).
Since these tools are made of metal, how can we use them?
They can be used and then re-sterilized in a flame repeatedly.
What else can grow on the media on which you want to grow your new culture?
Undesirable contaminants
What are examples of culture contaminants?
Molds; fungus; bacteria from your skin and hair
Nearly all forms of contamination are carried on what?
Microscopic dust particles
How can these particles affect experiments?
They make their way onto sterile surfaces when carelessly handled.
Is aseptic technique as pure as sterile technique?
No.
When a nurse or doctor cleans an infected wound, the site is considered contaminated already (since it is infected), so what technique is used?
ASEPTIC
What technique means you don’t have to scrub the patient’s skin with Betadine (antimicrobial scrub), use sterile gauze or wear a gown/face mask?
Aseptic
What technique is used if the patient is having a sterile surgery and you do have to scrub the patient’s skin while wearing a face mask and sterile gloves?
STERILE
What refers to being able to safely transfer organisms from one location to another, without spreading the organism to other locations, and without contaminating a pure culture we are trying to grow?
Aseptic technique
How long can you leave a culture dish open for when viewing colonies of organisms?
Never. Not even for a short amount of time.
What is the correct way to open a culture dish?
Keep the lid close to the dish, open it only as far and as long as is necessary. Keep it between your face and the agar surface.
What is the correct way to grab a loop or needle?
By the handle. It may be scalding hot from a previous student.
What is the correct distance to prepare equipment?
Within arm’s reach so you will not burn yourself while trying to inoculate tubes.
What should you label the tube or plate with?
Label plates with: Date, test type (Experiment #), lab period (Magrann), and your group name.
What is the correct way to prepare a Bunsen burner?
Adjust the flame so the yellow flame disappears and just the blue flame is visible with a blue cone.
What are the steps to flame a loop or needle?
- Hold it angled downward into the blue part of the flame.
- Start the flame at the loop.
- When the lower 1/3 of the instrument glows red, push the instrument further into the flame so the middle 1/3 is in the flame. When that glows red, push it in further so the upper 1/3 of the instrument can be flamed.
When does the loop/needle become sterile? How do you keep it sterile?
After the loop is fully glowing red.
Don’t let the instrument touch any surface until you are ready to obtain your inoculum.
What is an inoculum?
Scoop of bacteria
Can you flame a loop that is wet? Why or why not?
No. It will spatter, scattering bacteria with it.
How long should you let the instrument cool after flaming it?
30 seconds.
How do you make sure it is cool?
By touching it to the agar or liquid surface in your culture before you obtain your culture.
What happens if the instrument is too hot?
It will kill the culture on contact.
Can you wave the loop around to hasten cooling? Why or why not?
No. Don’t blow on it either. Either action could introduce bacterial contamination.