Lab Unit 1 Flashcards
(406 cards)
1
Q
What type of footwear is allowed in lab?
A
Closed toed shoes only
2
Q
Where must equipment go after you are finished with your lab assignment?
A
In the red tote box
3
Q
What must you do to the counter tops before and after lab?
A
Wipe them down with disinfectant.
4
Q
Where must you dispose of any hazardous materials including toothpicks, Petri dishes, gloves, etc?
A
The biohazard bag. Everything else goes into regular trash, including paper towels used to dry your hands.
5
Q
When putting away or retrieving glassware such as flasks, beakers, test tubes what must we first do?
A
Remove all grease marks.
6
Q
If there is a spill what are the steps we take to properly clean them?
A
Cover them with paper towels, saturate the spill with disinfectant for 10 mins.
7
Q
Where must we dispose what we used to clean it?
A
In the biohazard bag.
8
Q
What must be done before using your microscope?
A
Ensure that is has been put away correctly.
9
Q
What must be done after using your microscope?
A
Ensure that is has been put away correctly.
10
Q
Are you allowed to have food, drink, water bottles, or gum in lab?
A
No.
11
Q
Where is the first aid kit located?
A
In the cabinet by Andrea’s door.
12
Q
What are the four techniques of inoculation?
A
Streak plate
Streak for Isolation
Spread plate
Pour plate
13
Q
These two techniques are quantitative methods used to determine the number of bacteria in a sample.
A
Spread plate
Pour plate
14
Q
What is the spread plate technique?
A
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°.
15
Q
What is the pour plate technique?
A
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.
16
Q
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.
A
Streak plate method
17
Q
This technique is used to take a mixture of bacteria and separate them out into pure cultures to identify each organism.
A
Streak for isolation
18
Q
When using streak for isolation your petri dish should be divided into how many parts?
A
4 quadrants
19
Q
During a streak for isolation, after you finish EACH streak which way must you turn your plate?
A
Counter-clockwise
20
Q
Why is this technique so commonly used by doctors?
A
The physician needs to know what EXACTLY the organisms are so he/she may treat the patient accordingly.
21
Q
What does this method result in when the sample are taken?
A
Fewer organisms are dragged into each quadrant, so that the last quadrant does not overlap its colonies of organisms.
22
Q
What happens if the streak is successful?
A
In a few days you will see separate colonies in the last quadrant.
23
Q
What happens if it was unsuccessful?
A
Different looking colonies will be overlapping each other in the last quadrant.
24
Q
If your streak was unsuccessful, what could have been done incorrectly?
A
Too many organisms in the first quadrant and they too got dragged along.
25
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.
26
When do you use an inoculation needle?
If you need to take a pure colony from a plate. You need a needle.
27
What 3 things to look for in nutrient slant characteristics?
1. Is there turbidity (cloudiness)?
2. Is there growth only at the top (pellicle), only at the bottom (sediment), flakes in the middle (flocculent), or growth throughout (uniform turbidity)?
3. What color is it?
28
What are the 3 different types of plate characteristics?
1. Margins (look at the surface of the plate)
2. Texture (look at the surface of the plate)
3. Pigmentation (color of plate)
29
What are 6 types of colony margins?
1. Filiform (thread-like edge)
2. Arborescent (tree-like branches)
3. Beaded (starts smooth, breaks into individual colonies at top)
4. Effuse (edges are not clearly defined)
5. Rhizoid (root-like branches)
6. Echinulate (serrated edge)
30
What are the 4 types of colony texture?
1. Flay, dry (Bacillus megatarium)
2. Spreading (proteus, pseudomonas)
3. Crusty, waxy (mycobacterium)
4. Transparent
31
What to look for in pigmentation?
1. Yellow
2. White
3. Green
4. Etc.
32
What are the 3 different colony configuration types?
1. Round
2. Irregular
3. Rhizoid, arborized
33
What are the 4 different colony elevation types?
1. Convex
2. Flat
3. Raised
4. Umbonate (belly button protrusion)
34
To what are nutrients added to make media for bacteria cultures?
Agar (seaweed product)
35
What type of nutrient medium does not solidify?
A broth
36
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.
37
What happens after inoculation?
The plate or tube is usually incubated for at least 24 hours to encourage growth of the sample.
38
What two ways are culture media classified?
Consistency and contents
39
What are 4 media classifications based on their contents?
1. All-purpose
2. Selective
3. Enriched
4. Differential
40
What does consistency refers to?
A liquid, solid, or semi-solid.
41
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.
42
What organisms are not supported by nutrient agar (NA)?
Fastidious (hard to grow) organisms.
43
What are the 3 types of isolation media?
1. Selective media
2. Enrichment media
3. Differential media
44
What is selective media?
Selects for a particular type of organism to grow.
45
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.
46
What is an example of selective media?
Sabouraud’s Dextrose Agar (SDA)
47
What does SDA contain?
It has a high sugar content and an acidic pH.
48
What is the classification of SDA media?
Selective, or Isolation media
49
Why is that?
It isolates molds and yeasts, which do well in high sugar content, whereas bacteria inhibited.
50
What natural substance is a good a bacterial preservative?
Sugar
51
Do jams, jellies, and preserves grow bacteria?
No
52
Are molds aerobic or anaerobic?
Aerobic
53
How do molds get onto our food?
Microscopic air-borne spores.
54
Should you throw out all of the food when you suspect mold? Why?
Yes. Some species produce toxins that cause liver cancer.
55
What pH do molds and yeasts prefer?
Acidic
56
What media was originally designed to isolate dermatophytes?
SDA (Sabouraud’s Dextrose Media)
57
What is a fungus that can infect skin, nails, hair, and are opportunistic pathogens?
Dermatophytes
58
What produces ringworm, especially athlete’s foot?
Dermatophytes
59
What microbes are known for causing infections in women, diabetic, and cancer patients?
Yeasts
60
Why are diabetics more prone to infections?
They are immunocompromised due to their high glucose levels.
61
How do you isolate a fungus from the skin?
Scrape off a little skin and place in SDA
62
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
63
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
64
When using differential media, how can you tell which group of bacteria there are?
By the color the plate turns
65
What type of media has added nutrients such as vitamins and amino acids to enhance the growth of desired bacteria?
Enriched media
66
What type of media is used to grow bacteria which are hard to grow, known as fastidious organisms?
Enriched media
67
Are most pathogenic organisms fastidious?
Yes
68
What is an example of enriched media?
Blood agar
69
What media is the same as NA, but has no agar?
Broth
70
What are made exactly as NA except poured into tubes instead of plates?
Slants
71
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).
72
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.
73
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.
74
The basic frame of the microscope consists of what 4 parts?
Base, stage, arm, and body tube
75
What raises and lowers the condenser?
The substage adjustment knob
76
When you are done with the microscope, leave the condenser in the highest position. This is called...
Racking up the condenser
77
What is the eye piece called?
Ocular
78
What magnifies ten times and focuses the image on the retina?
Ocular
79
How do you clean the oculars?
Squirt alcohol onto lens paper and wipe, then dry with lens paper.
80
Which ocular has a diopter adjustment ring?
The left ocular
81
What is the purpose of the diopter adjustment ring?
To allow the left eye to be focused independently of the right eye.
82
What revolving piece holds the objectives?
The nosepiece
83
What is the smallest objective lens?
The scanning (red ring) objective
84
What is the magnification of the scanning objective?
4x
85
How do you find total magnification?
Multiply the ocular magnification by the magnification of the objective.
86
What is the total magnification of the scanning objective?
40x
87
What is the total magnification of the low power objective?
100x
88
What is the total magnification of the high dry objective?
400x
89
What is the total magnification of the oil immersion objective?
1000x
90
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
91
What is the total magnification of a microscope that has a 10x ocular, when using the low power lens?
10 x 10 = 100x
92
What is the total magnification of a microscope that has a 10x ocular, when using the high dry lens?
10 x 40 = 400x
93
What is the total magnification of a microscope that has a 10x ocular, when using the oil immersion lens?
10 x 100 = 1000x
94
What is the working distance?
The distance between the stage and the objective lens.
95
What does parfocal mean?
Once you are focused with the scanning objective, you are focused with all of the objectives.
96
What takes light from the lamp and makes the rays into a point on the slide?
The condenser
97
What opens and closes like the iris in your eye (pupil)?
The iris diaphragm lever
98
Why does the iris diaphragm lever open and close?
To regulate the amount of light allowed in.
99
When changing from low to high power, what needs to be done to the iris diaphragm?
It needs to be opened.
100
What is required for increasing magnification?
MORE LIGHT
101
What are the four things the iris does?
Brightness
Contrast
Depth of field
Resolution
102
When the iris is open, what happens to contrast?
Decreases
103
When the iris is open, what is in focus?
Only the foreground
104
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.
105
What is the working distance?
The length between the tip of the objective lens and the stage
106
Which objective lens do you start with?
Lowest power
107
As you increase magnification, what happens to working distance?
Decreases
108
Why does this trend occur?
Because the objectives are taller as you go up in magnification.
109
Which objective has the largest field of view?
the scanning objective
110
Which objective has the greatest working distance?
the scanning objective
111
Which objective has the smallest field of view?
oil immersion lens
112
Which objective has the shortest working distance?
oil immersion lens
113
What is the substage adjustment knob used for?
Moves condenser up and down (when down, there is poor resolution).
114
What two things affect the resolving power of a microscope?
The numerical aperture of the lenses.
| The wavelength of light.
115
What is the diopter ring used for?
Allows left eye to focus independently.
116
What organisms are so small, so they always need 1000x and oil to be seen?
Bacteria
117
What are three basic shapes of bacteria?
Spiral
Cocci (singular is coccus)
Bacilli (singular is bacillus; also known as rods)
118
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.
119
True or false: When putting away the microscope, the AC (power) cord is wrapped loosely behind the scope.
True
120
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.
121
True or false: When putting away the microscope, the condenser should be racked up (placed in its highest position).
True
122
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
123
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.
124
True or false: When putting away the microscope, the stage should be racked down.
True
125
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.
126
When putting away the microscope, what needs to be cleaned from the stage and the microscope in general?
Oil and fingerprints
127
When putting away the microscope, what needs to be used to wipe the ocular lens?
Lens paper only
128
When putting away the microscope, where should the eyepieces face?
The blue side of the scope
129
When putting away the microscope, what should be done to the screw?
Tighten it a bit.
130
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.
131
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
132
How are these microbes killed?
Autoclaving (steam heat sterilization)
133
How should these microbes be handled?
By aseptic technique and with safety equipment under the hood.
134
What gear do these microbes require?
A space suit type of gear.
135
What type of air pressure does a room dealing with these microbes require?
Negative pressure
136
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.
137
How often is the air in this room need to be circulated?
About every eight minutes
138
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).
139
Since these tools are made of metal, how can we use them?
They can be used and then re-sterilized in a flame repeatedly.
140
What else can grow on the media on which you want to grow your new culture?
Undesirable contaminants
141
What are examples of culture contaminants?
Molds; fungus; bacteria from your skin and hair
142
Nearly all forms of contamination are carried on what?
Microscopic dust particles
143
How can these particles affect experiments?
They make their way onto sterile surfaces when carelessly handled.
144
Is aseptic technique as pure as sterile technique?
No.
145
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
146
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
147
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
148
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
149
How long can you leave a culture dish open for when viewing colonies of organisms?
Never. Not even for a short amount of time.
150
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.
151
What is the correct way to grab a loop or needle?
By the handle. It may be scalding hot from a previous student.
152
What is the correct distance to prepare equipment?
Within arm's reach so you will not burn yourself while trying to inoculate tubes.
153
What should you label the tube or plate with?
Label plates with: Date, test type (Experiment #), lab period (Magrann), and your group name.
154
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.
155
What are the steps to flame a loop or needle?
1. Hold it angled downward into the blue part of the flame.
2. Start the flame at the loop.
3. 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.
156
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.
157
What is an inoculum?
Scoop of bacteria
158
Can you flame a loop that is wet? Why or why not?
No. It will spatter, scattering bacteria with it.
159
How long should you let the instrument cool after flaming it?
30 seconds.
160
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.
161
What happens if the instrument is too hot?
It will kill the culture on contact.
162
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.
163
What is the correct way to hold the tube that contains the culture and the sterile loop?
Hold the tube by the base in your NON-DOMINANT hand. Hold the sterile loop in your DOMINANT hand.
164
What is the correct way to take off the top of the culture tube?
Take the top off with your DOMINANT hand, between your pinky and ring finger.
165
Can you place the lid on another surface?
NO. You must hold the lid in your hand during the transfer.
166
How do you make sure that no contaminants get into the tube during transfer?
Make sure the open side of the lid is facing downward. That way, no contaminants can float down into it.
167
Which hand should be holding the lids and the sterile loops?
DOMINANT HAND
168
Which hand should be holding the culture tube?
NON DOMINANT HAND
169
What is the correct way to flame the tube?
Pass the neck of the glass culture tube through a flame (2 quick, back-and-forth strokes), with the container at a 45 degree angle toward the flame. Use the blue part of the flame.
170
What hand should be holding the tube while flaming it?
NON DOMINANT
171
What hand should hold the sterile, cooled loop?
DOMINANT
172
How should you obtain your inoculum from the culture tube?
Stick the sterile, cooled loop or needle into the culture tube, obtain your culture inoculum, and then withdraw the loop or needle.
173
Is it necessary to flame the tube again after obtaining your inoculum?
Yes. Place the lid back on after doing so.
174
What should you be aware of while re-flaming the tube?
Pay close attention to the pure culture inoculum being held by your dominant hand. Don't let it accidentally touch any surface or you will have to flame it and start over.
175
What does a tube usually contain?
Nutrient Broth or solid agar.
176
Which instrument should you use to obtain an inoculum from a tube?
Loop
177
How should you hold the loop while obtaining an inoculum from a tube?
Hold it like a pencil in your dominant hand.
178
Should you shake a tube of culture broth before removing a sample? Why or why not?
Yes, ALWAYS shake the tube because it disperses bacteria that might otherwise have sunk to the bottom of the tube.
179
How do you obtain the inoclum if the tube contains solid agar?
Gently scrape the top of the loop across the surface of the culture in the tube while being careful not to dig into the agar.
180
How do you position the loop in relation to the agar?
Keep the loop parallel to the agar surface to prevent digging into the medium.
181
How do you obtain a large inoculum from a broth culture?
Use a sterile disposable pipette.
182
What is the correct way to obtain an inoculum from a Petri dish with a LOOP?
Scrape the top of the loop across the surface of the culture, being careful to keep the loop parallel to the agar surface to prevent digging into the medium.
Remember: Don't take the lid off completely, just lift it a little to obtain the sample.
183
What is the correct way to obtain an incoulum from a Petri dish with a NEEDLE?
Just touch the tip of the needle to a single colony, being careful not to dig into the medium. Don't pick up more than one colony.
184
When a Petri dish contains multiple types of bacteria and you want to subculture one pure colony to run tests on it for identification, would you use a loop or needle to obtain an inoculum?
Needle.
185
What are the steps to inoculate a Petri dish?
1. Pick up the lid with your NON DOMINANT hand, and only raise it a little, keeping the lid face down and directly above the Petri dish.
2. The Inoculum on the loop should still be in your DOMINANT hand.
3. Streak the plate with the loop or needle, being careful not to touch the outer surface of the Petri dish. (Since both hands are occupied, the Petri dish base must be left on the table).
4. Replace the lid, re-flame the loop or needle being careful to put it down safely without burning anything.
186
What are the steps to inoculate a tube?
1. Pick up the tube with your NON-DOMINANT hand (Tube Hand).
2. Take off the lid with the two little fingers of your DOMINANT hand (Loop Hand).
3. Flame the glass neck
4. Inoculate
5. Flame the neck again
6. Set the tube down in the rack
7. Replace the lid
8. Re-flame the loop or needle.
187
How do you inoculate a broth in a tube?
1. Place the loop in the broth
| 2. Knock the loop back and forth across the sides of the tube with the loop immersed in the broth.
188
How do you inoculate an agar tube?
There are several techniques (streak, swab, etc). When transferring cultures, hold the tubes by the bas of the tube in your NON DOMINANT hand and the sterile loop in your DOMINANT hand.
189
How do you inoculate onto an agar slant with a loop?
Place the loop with bacteria into the slant tube, all the way down to the bottom of the slant. You may either just bring the loop straight up the slant, or you may zig-zag the streak as your bring it up.
190
How do you inoculate onto an agar slant with a needle?
Stab the inoculum down to the bottom of the agar in a clean, straight stroke. Pull the needle out of the same hold you created win you stabbed it. (Sometimes you also streak the top of the surface with a zig-zag, check directions for each experiment).
191
Proper order steps for staining are?
1. place organism on slide
2. air dry
3. heat fix
4. stain
192
What is the purpose of Heat Fixing?
To attach the cells (or bacteria) to the slide and kill the microbes.
193
What does Heat Fixing do?
Shrinks the cells and causes proteins in the cells to become like glue. (easier seen/stained)
194
What is a Stain?
A dye that is made of salt w/ a colored ion (called a chromophore)
195
What is an ion with a positive charge called?
Cation
196
What is an ion with a negative charge called?
Anion
197
A stain with a cation has what pH?
Creates basic dye (pH higher than 7)
198
A stain with an anion has what pH?
Creates an acidic dye (pH lower than 7)
199
What is the type of stain that we will mainly be using in lab?
Basic dye (cation chromophore)
200
What is a negative stain acidic or basic?
Acidic
201
Give 3 examples of a negative stain.
Nigrosin
India Ink
Congo Red
202
What does a slide look like when a negative stain is used?
The background is stained instead of the cells.
203
What dye is used when you want to measure the size of cells and to clearly see the shape of an organism?
Negative stain
204
Do we heat fix a slide when using a negative stain? Why not?
No, you simply mix the cells in the dye and spread it out thinly. Because there is no heat fixation, the cells don’t shrink and you can see the natural size of the cells.
205
What do you measure cells by using a tiny ruler in the eyepiece of a microscope called?
Ocular micrometer
206
What are the 4 types of stains we will use?
1. Differential Stain
2. Special Stains
3. Simple Stain
4. Negative Stain
207
What are differential stains? Give 2 examples.
Several stains are used in the procedure.
| Examples are Gram stain and Acid-Fast stain
208
What are differential stains used for?
To find out what category of bacteria are present.
209
What are 3 types of Special Stains?
Endospore stain
Capsule stain
Flagella stain
210
What is a simple stain? Give one example.
Only one stain is used.
| An example is methylene blue.
211
What are the 4 steps in a Gram stain?
1. Primary Stain
2. Mordant
3. Decolorizer
4. Counterstain
212
What is the Primary Stain in a Gram stain procedure?
Crystal violet.
| This is the first stain used.
213
What is the Mordant in a Gram stain procedure? What is a mordant?
Iodine
The mordant is what allows the primary stain to react chemically w/ the cell. It forms a complex violet and keeps it from being washed out by the alcohol.
214
What is the Decolorizer in a Gram stain procedure?
Alcohol or Acetone
This removes the primary stain (decolorizes)
from the Gram negative cells. It now has no color.
215
What is the Counterstain in a Gram stain procedure?
Safranin
| This is a red color that stains the Gram negative cells after they have been decolorized.
216
Purpose of a Gram Stain?
Used to distinguished between:
- Gram Positive bacteria (will look purple because they retain the primary stain after the decolorizer)
- Gram Negative bacteria (will look pink from safranin because they were decolorized)
- Since all bacteria are either gram +/- this stain is the first thing used to determine what type of bacteria is present in the specimen.
- This helps us figure out what organism we are dealing w/. the results are recorded as Gram +/-
217
Give an example of an Acid Fast Stain. How are results recorded?
Example is ZIEHL-NEELSEN STAIN
| Results are recorded as acid-fast or non acid-fast.
218
What colors are acid fast and non acid fast when using the Ziehl-Neelsen stain?
Acid fast bacteria look pink and non acid fast look blue.
219
If bacterium is positive for the acid fast stain, what does it mean?
It has mycolic acid in the cell wall.
220
What is Mycolic Acid?
Is a very waxy substance that resists Gram Stains. The heat in this procedure will melt down the wax in the cell wall to allow the stain to get in.
221
Name 2 pathogenic organisms that are acid fast and the diseases they cause.
1. Mycobacterium (tuberculosis and leprosy)
| 2. Nocardia (pneumonia)
222
What is the primary stain in an acid-fast procedure?
Carbol Fuchsin (purplish pink)
223
What is the mordant in an acid-fast procedure?
Heat
224
What is the decolorizer in an acid-fast procedure?
ACID alcohol
225
What is the counterstain in an acid-fast procedure?
Methylene blue
226
What are Opportunistic Infections?
Infections caused by organisms that usually do not cause disease in a person w/ a healthy immune system, but can affect people w/ a poorly functioning or suppressed immune system. They need an “opportunity” to infect a person.
227
Examples of Immunocompromised patients are?
1. Elderly people or infants
2. Aids/HIV- infection
3. Immunocompromised agents for organ transplant recipients.
4. Chemo patients
5. Malnutrition
6. Medicines (some antibiotics)
7. Medical procedures (surgeries especially implanted joint replacement or internal fixation hardware such as screws and plates for broken bone)
228
What type of stain is used to view endospores, capsules or flagella?
SPECIAL STAINS are used to view endospores, capsules, or flagella.
229
What are three special stains?
1. Endospore Stain
2. Capsule Stain
3. Flagella Stain
230
How do some bacteria adapt when the environment becomes too harsh?
When the environment becomes too harsh to survive, some bacteria have the ability to eliminate all their cytoplasm and condense all their essential DNA and organelles into an endospore, which is resistance to chemicals, heat, and dry environments. When the environment improves, they can re-establish themselves.
231
Is an endospore metabolically active or inactive?
Endospores are metabolically inactive.
232
Malachite green
PRIMARY STAIN
233
Heat (allows dye to penetrate the endospore)
MORDANT
234
Water
DECOLORIZER
235
Safranin
COUNTERSTAIN
236
What kills an endospore?
Only sterilization can kill an endospore.
237
Endospores are only produced by what type of bacteria, and where are they found?
Gram positive Bacillus bacteria, which are found in the soil
238
What are two examples of bacteria that make endospores?
Bacillus megatarium (non-pathogenic) & Clostridium (tetanus and botulism)
239
```
ENDOSPORE STAIN:
Name the PRIMARY STAIN.
Name the MORDANT.
Name the DECOLORIZER.
Name the COUNTERSTAIN.
```
PRIMARY STAIN: Malachite green
MORDANT: Heat
DECOLORIZER: Water
COUNTERSTAIN: Safranin
240
What is the purpose of a capsule for bacteria?
Resists phagocytosis (being eaten by our white blood cells)
241
What stain is used for bacteria that have a capsule?
Capsule Stain
242
Name one example of a bacterium that has a capsule.
An example is Streptococcus pneumoniae.
243
What effect(s) does a Capsule Stain have on the background; and the capsule itself?
This stain colors the background.
| The capsule remains clear.
244
What is a flagellum, and how is it used by bacteria?
A flagellum is a whip-like tail used to help bacteria move.
245
Is the flagellum easily seen using ordinary stains?
NO. It is so thin it is not easily seen with ordinary stains.
246
What special stain is used to view flagella?
Flagellar Stain
247
Name one example of a Simple Stain.
Methylene Blue
248
When are Simple Stains used?
For cheek cells or bacterial colonies
249
What type of stain is NIGROSIN?
NEGROSIN is a Negative Stain.
250
Is it necessary to heat-fix Nigrosin? Why or why not?
There is no need to heat-fix Nigrosin because the stain makes the bacteria stick to the slide. Just let it air dry.
251
What is an ion with a negative charge called?
An ANION
252
What is a negative stain?
A negative stain is one that contains anions on the chromophore (color portion of the dye).
253
Is the color portion of the dye a positive or negative charge? Acidic or not acidic?
The color portion of the dye has a negative charge. It will also be acidic.
254
Are bacteria cells positively or negatively charged?
Bacteria cells have a negative charge.
255
What affect does the charge of a bacteria cell have on the chromophore (dye)?
The bacteria cells repel the chromophore (dye)
256
When using a negative stain, will the cell have COLOR or NO COLOR?
The cell will have NO COLOR.
257
When using a negative stain, is the background STAINED or NOT STAINED?
Only the background is STAINED.
258
What is the purpose of using a Negative Stain?
The purpose of using a negative stain is to determine the size and shape (bacilli, cocci, spirals) of an organism and their arrangement (staphylo, tetrads, strepto, etc).
259
What are the three types of negative stains?
The three types of negative stains are:
1. Nigrosin
2. India ink
3. Congo red
260
What is the most common type of stain?
Gram Stain
261
What is the first step in identifying an organism?
A GRAM STAIN is the first step in identifying an organism. It does not identify the organism, but it is the first step.
262
What are the reactions of each stain type different?
The reactions of stains are different because of differences in the chemistry of the cell wall.
263
What is difference between the cell wall of a Gram positive bacteria and the cell wall of a Gram negative bacteria?
Unlike Gram positive bacteria, the cell wall of a gram negative organism has many lipids in the cell wall:
1. LPS – lipopolysaccharide
2. LP – lipoproteins
3. PL – phospholipids
264
Name the three characteristics of GRAM POSITIVE.
1. Purple
2. Thick peptidoglycan
3. Very little lipid
265
Name the three characteristics of GRAM NEGATIVE.
1. Pink
2. Thin peptidoglycan
3. Lots of lipid (in the outer membrane)
266
What is the GRAM STAIN used for; and what does it look like?
The Gram stain is used to distinguish between Gram positive bacteria (will look violet because they are not decolorized) and Gram negative bacteria (will look pink from the safranin because they were decolorized).
267
What are the four steps in the Gram Stain Procedure?
First, prepare the sample on a slide, air dry and heat fix.
1. PRIMARY STAIN
2. MORDANT
3. DECOLORIZER
4. COUNTERSTAIN
268
What stain is used first?
PRIMARY STAIN
269
What is a mordant?
A MORDANT causes the primary stain to form a complex with it to chemically react with the peptidoglycan in the cell wall. It keeps the crystal violet from being washed out by the alcohol in the next step.
270
What is used as the mordant in the Gram Stain procedure?
Gram’s Iodine
271
What would happen without Iodine?
The crystal violet stain would wash away and Gram positive organisms would look pink.
272
What removes the primary stain from the Gram negative cells? (decolorizes them)?
Alcohol (ETOH)
273
What is most critical step because the alcohol must be left on for just the right amount of time?
The decolorizer step (alcohol wash)
274
What is used as the counterstain in the Gram stain procedure?
Safranin
275
What would happen if you forget to use Gram’s iodine?
Both Gram positive and Gram negative cells would be pink
276
What would happen if you over-decolorize?
Both Gram positive and Gram negative cells would be pink
277
What would happen if you use no alcohol?
Both Gram positive and Gram negative cells would be purple
278
What would happen if you use an old culture?
Gram negative cells might be purple, Gram + cells might be pink or purple and pink because some PG has broken down
279
What are some reasons why you might get a Gram-positive culture show up with both purple and pink cells that are all the same size?
1. The culture is too old (more than 24 hours). Some cells have died, and the peptidoglycan breaks apart, so it appears Gram negative.
2. Too much decolorizer was used.
3. The sample smear was too thick and the stain did not get through to all the cells.
280
Suppose you look at a Gram stain of staphylococcus and you see staphylo, diplo, singles, and tetrads on your slide. They are all purple and they are all the same size. Is the culture contaminated?
No, it is probably still pure because the cells are all the same size. The clusters just broke apart during the preparation, or you are seeing a new cell in the process of dividing into a cluster.
281
What does it mean when you see both purple and pink cells of different shapes?
That it is not a pure culture
282
Name three types of isolation media
MANNITOL SALT AGAR (MSA)
EOSIN-METHYLENE BLUE (EMB) agar
BLOOD AGAR
283
What media contains a lot of salt? Only Gram-positive organisms can tolerate this.
MANNITOL SALT AGAR (MSA)
284
What are the only organisms that grow on MSA?
Gram-positive organisms
285
What media contains two dyes that inhibit Gram-positive bacteria, but are attractive to Gram negative bacteria?
EOSIN -METHYLENE BLUE (EMB) Agar
286
MSA and EMB agar are considered what type of media?
Both are Selective Media, and MSA is also Differential Media.
287
Why are MSA and EMB considered Selective Media?
Because only certain types of bacteria will grow on them.
288
What plate contains a sugar called mannitol?
MSA
289
What is the waste product when sugar is used by bacteria?
Acid
290
What is methyl red?
It is a red colored pH indicator that will turn yellow if acids are present. That means the organism ferments the sugar in the medium.
291
What type of media is MSA?
Selective because of salt (only Gram positive organisms can grow on it), and differential because of mannitol (only mannitol fermenters turn it yellow)
292
What type of media is blood agar?
ENRICHED MEDIA
293
When do we use blood agar?
To grow organisms that are fastidious (picky eaters), such as pathogenic bacteria
294
What is an example of a fastidious organism that needs to be grown on blood agar?
Streptococcus pyogenes (causes strep throat)
295
What are the three types of hemolysis patterns that can be seen on blood agar?
```
alpha hemolysis (blood turns green due to partial hemolysis of the red blood cells)
beta hemolysis (blood turns clear due to total hemolysys of the red blood cells)
gamma hemolysis (no change in blood color due to lack of hemolysis)
```
296
What machine is used to measure the turbidity of living and dead organisms in a sample?
Spectrophotometer
297
Is using a spectrophotometer to measure turbidity a direct or indirect estimate of cell density?
Indirect
298
What is the formula for calculating concentration of an organism in a water sample that was diluted?
Colony numbers x Dilution Factor
299
A standard plate count (SPC) is a direct method of enumeration of bacteria that measures what?
Measures living organisms per ml, plated and count the colonies that grow.
300
Before a SPC is done what else has to be performed to ensure proper results?
A serial dilution of the original broth culture.
301
Which machine sends a beam of light through a tube of liquid sample and reads how much light comes out?
Spectrophotometer
302
Are transmission and absorbance directly or inversely related?
Inversely
303
If a sample is not clear because it contains organisms what will the transmission percentage be?
Less than 100%
304
What happens to the light that is not transmitted when measuring turbidity?
It is absorbed.
305
If 70% of light is transmitted out of 100% how much is absorbed?
30% is absorbed
306
Special test tubes called cuvettes which are known are optically pure glass differ from regular glass test tubes in what way?
They will not absorb light.
307
What kind of broth is the sample in and what color is it?
Nutrient broth a brown color
308
When placing a cuvette on the spectrophotometer and setting the machine to zero what are you doing?
Preparing a “blank”
309
If we do not blank and set the machine to zero what will happen to our readings?
They will be lower than they should be.
310
A transmission reading should be between what?
1-99%
311
After the machine has been “blanked” and zeroed and the first sample has been read what is the next step for measuring your next sample?
Nothing the machine does not need to be blanked or zeroed anymore.
312
How is turbidity recorded as?
Samples optical density
313
A measurement of the transmission of light passing through a liquid sample is known as what?
Optical Density
314
If a sample is cloudier aka more turbid what will the transmission be?
The transmission will be lower.
315
Low transmission and high optical density means what?
A lot of bacteria in the sample
316
High transmission and low optical density means what?
Few bacteria in the sample
317
Optical density and transmission are considered to be?
Inversely related.
318
To make a standard plate count what needs to be done first?
Dilute the original sample
319
What is considered the number of colonies within our ability to count?
30-300
320
A dilution series is done to see what?
Which plate grows the number of colonies within ability to count.
321
To calculate the number of living organisms in the original sample what needs to be done?
Multiply number of colonies by dilution factor
322
50 colonies were counted on a plate with 1:1000 dilution. What is total number of organisms in the original solution?
50x 1000= 50,000 use abbreviations for organisms and milliliters (org/ml)
5.0x 10 to the 4th power is (10,000) This equals the number of living of organisms in the original sample.
323
What does pH measure the concentration of?
Hydrogen ions (H+)
324
What makes a substance more acidic, more H+ ions or less H+ ions?
The more H+ ions, the more acidic the substance is, which means it has a low pH (below 7).
325
What makes a substance more basic (or alkaline), more H+ ions or less H+ ions?
The less H+ ions, the more basic (or alkaline) the substance is, which means it has a high pH (above 7).
326
What is the pH of neutral substances, like water and much of our body fluids?
pH 7
327
What does the pH scale run from?
pH 2 (acidic) to pH 14 (base)
328
What does each unit change in pH represent?
Each unit change in pH represents a 10 fold difference in H+ ions
329
Do acids have more or less H+ ions?
Acids have more H+ ions
330
When you go from a high pH to a low pH, is there an increase or decrease in H+?
Increase
331
When you go from a low pH to a high pH, is there an increase or decrease in H+ ions?
Decrease
332
What does a change from pH 3 to pH 4 represent?
10x decrease in H+ ions
333
What does a change from pH 3 to pH 2 represent?
10x increase in H+ ions
334
What does a change from pH 3 to pH 5 represent?
100x decrease in H+ ions
335
What does a change from pH 3 to pH 6 represent?
1000x decrease in H+ ions
336
What does a change from pH 10 to pH 2 represent?
100,000,000x increase in H+ ions
| Subtract 2 from 10 and get 8. That is the number of zeros to place after the one
337
What does a change from pH 2 to pH 10 represent?
100,000,000x decrease in H+ ions
338
What happens when an organism is placed in a substance that is suboptimal (meaning less than the best for them)?
The proteins in the cell walls of the vegetative cells can become denatured (damaged)
339
What are organisms that grow best at a pH 2 and pH 4 called?
Acidophiles
340
What are organisms that grow best at pH 7 called?
Neutrilophiles
341
What are organisms that grow best at pH 10-12 called?
Alkaliniphiles
342
What is an opportunistic pathogen?
It only causes infection if it has an opportunity to invade.
343
What is an example of an alkaliniphile?
Alcaligenes faecalis (an opportunistic pathogen) This bacterium degrades urea to make ammonia, which increases the pH.
344
What disease is caused by Alcaligenes faecalis?
This organism causes urinary bladder infections, so a patient with a catheter might be at risk.
345
What is an example of an acidophile?
Saccharomyces cerevisiae
346
What is Saccharomyces cerevisiae?
Yeast which is used to make beer. Many yeasts and fungi use fermentation as a metabolic pathway, and acids are produced in the process.
347
What is an example of a neutrophile?
Staph aureus
348
What is Staph aureus?
A resident organism (lives on our skin without causing harm) which is an opportunistic pathogen. If we get a cut, it can take the opportunity to invade and cause the wound to become infected.
349
Which organism would have a high OD at pH 2?
Acidophile
350
Which organism would have a high OD at pH 12?
Alkalinophile
351
Which organism would have a high OD at pH 7?
Neutrophile
352
What does the replication area in DNA of bacteria contain?
Two adjacent thiamines
353
How does UV light kill bacteria?
UV light causes a bond to form between the two thiamines, and stops DNA replication, which is the cause of death in the organism.
354
What would happen if an organism could produce endospores?
It could survive chemicals, heat, and UV light exposure for a longer time.
355
What are the only bacteria that make endospores? What is an example?
Gram positive rods are the only bacteria that make endospores. An example is Bacillus megatarium.
356
What can you do to see how long it takes to kill each type of bacteria with UV light?
You could design an experiment to expose plates of two organisms to UV light for various periods to see how long it takes to kill each type of bacteria.
357
How long would you expose Staph aureus (Gram positive cocci) to UV light?
It would no longer grow after being exposed to UV light for just a few seconds.
358
How long would you expose Bacillis (Gram positive rod) to UV light?
It would have to be exposed to UV light for a longer time (few minutes) before it will stop growing since it can produce endospores, so it survives longer.
359
What is the formula to measure resistance to UV light?
Time to kill Bacillis ÷ time needed to kill Staph
Suppose the more resistant organism took 30 minutes to kill, and the other organism took 10 minutes to kill. 30 ÷ 10 = 3. That means organism 1 is 3x more resistant to UV light than organism 2.
360
Why do we sometimes incubate plates in a heater or cooler, and sometimes we leave them in room temperature?
Organisms have different temperature requirements.
361
What temperature do human pathogenic organisms prefer?
Body temperature (37 degrees Celsius)
362
What temperature do non-pathogenic organisms prefer?
Room temperature (25 degrees Celsius)
363
Are bacteria more or less heat resistant than other forms of life?
More heat resistant
364
The types of pathogenic bacteria that have flagella for motility often exist in what two forms?
1. They have a flagellum when they are outside of a human body.
2. They lose their flagella when they enter the body. The warmer temperature causes the flagellum to drop off.
365
When testing for motility of an organism, what temperature do you incubate at?
You have to incubate at room temperature or they will lose their flagella and test as non-motile.
366
Generally, if heat is applied, what happens to microbes?
They are killed
367
If cold temperatures are applied, what happens to microbes?
Their growth is inhibited, but they are not killed
368
What temperature do Psychrophiles prefer?
Freezing cold temperatures
369
What temperature do Mesophiles prefer?
Anything from room temperature to body temperature (25-40 degrees Celsius)
370
What temperature do Thermophiles like?
They like it quite hot (45-65 degrees Celsius)
371
What temperature do Hyperthermophiles prefer?
Extreme heat, such as found in volcanoes
372
What are most human pathogenic bacteria?
Mesophiles
373
What is an antiseptic used on?
Living tissue, not inanimate objects
374
What are the two kinds of antiseptics?
Topical (like Bactine)
| Oral (like Scope and Listerine)
375
Disinfectants are chemicals that are used on what?
Inanimate objects
376
For antiseptics and disinfectants, products with larger or smaller zones of inhibition are most effective?
The largest zones are the most effective. (NOTE: this is NOT the case with antimicrobials, where the largest zone is not necessarily the best).
377
Which agent is one that causes temporary inhibition of growth of bacteria?
Bacteriostatic
378
Which agent kills bacteria?
Bacteriocidal
379
What is a chemical used on NON-living tissue to decrease the number of microbes?
Disinfectant
380
What is a chemical used on living tissue to decrease the number of microbes?
Antiseptic
381
This infection is one that is acquired after a patient is admitted to a hospital.
Nosocomial
382
How can nosocomial infections be avoided?
By proper hand washing between patients.
383
These organisms are on our hands for a short while. We pick these up by touching objects, etc. Washing with soap and water, even for a short time will remove some of them.
Transient Organisms
384
These organisms stay on us, even after we wash with soap and water.
Resident Organisms
385
Do resident organisms cause us disease?
No, not unless there is a break in the skin.
386
Are your resident organisms harmful to another person?
They might be.
387
Even when washing your hands with soap and water why are only some resident organisms removed and not many, if at all?
Because it is impossible to sterilize living tissues.
388
What are the two kinds of antimicrobials?
Narrow Spectrum and Broad Spectrum.
389
Which antimicrobials are effective against Gram positive organisms?
Narrow Spectrum Antimicrobials.
390
How is this process done?
Their mechanism of action is a cell wall inhibitor.
391
Which antimicrobials kill both Gram positive and Gram negative organisms?
Broad Spectrum Antimicrobials.
392
How is this process done?
The part of the cell that they damage is present in both types of organisms
393
Why do you need to be careful with broad spectrum antimicrobials?
Because if there are a lot of Gram negative bacteria in the blood stream and you kill them, their endotoxins will be released into the blood stream, which can be deadly to the patients.
394
What is the zone of inhibition?
The clear zone around the disc, which has no bacterial growth.
395
Each laboratory uses the same media and antibiotic discs, and the size of each disc is always the same. Why is this?
To make experiments standardized and you can use an antibiotic sensitivity table (Kirby-Bauer Char) to evaluate your results.
396
Discs are the same size, how do you measure the zone inhibition?
You measure from the edge of the disc to the edge of the clear zone.
397
How do you measure in an antiseptic experiment?
You measure from one edge of the clear zone to the other.
398
The zone of inhibition determines the effectiveness of the antimicrobials ONLY when checked against what?
The Kirby-Bauer chart.
399
If one zone of inhibition of an antibiotic disc is larger than another, does that mean that it is a more effective antibiotic?
No.
400
Suppose you measure all the zones of inhibition on your plate, and the largest one measures 20 mm. Is that the antibiotic that is most effective?
NO! We cannot answer that question until we check the chart.
401
According to this sample chart, if the zone of inhibition for your Cephadroxil disc was 20mm, the organism is resistant to that antibiotic. Should you prescribe Cephadroxil to that patient?
You should NOT prescribe Cephadroxil to that patient.
402
Why is this?
Because the zone for Cephadroxil needs to be 25mm or more to be effective.
403
What implies clinical efficacy only in body sites where the drugs are physiologically concentrated or when a higher than normal dosage of a drug can be used?
Intermediate Sensitivity
404
Choosing an antibiotic in what category is the best choice?
An antibiotic that is in the sensitive category.
405
If the first treatment option is not possible (because there are no such drugs for that organism, or the patient is allergic to the drug), the second treatment option is what?
Choose two antibiotics in the “intermediate” category is the next best option. The patient must take both of them (combination therapy), because they have a synergistic effect.
406
Why is it the next best option to prescribe two intermediate sensitivity antibiotics to someone who is allergic to the best antibiotic?
Because they give a synergist effect. One might inhibit protein synthesis, while the other interferes with the cell wall structure. Alone, they cannot kill the organism well, but together they are effective.
