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1

Wear proper clothing

wear appropriate shoes
no open-toe shoes or sandals
no easily flammable garments
long hair tied back
clothes must reach knees (no shorts/skirts)

2

Know location, operational details of all laboratory emergency safety equipment, disposal procedures, and evacuation routes.

eye wash in the front and at every sink
shower in the front
dispose of glass in ceramic jar, biohazards in red box, trash in trash

3

Eating, drinking, or use of tobacco products is strictly prohibited in science labs.

Food & drinks in backpacks

4

Do not work in the laboratory without the permission of and presence of responsible staff.

Do not work in the laboratory without the permission of and presence of responsible staff.

5

Perform only assigned laboratory activities, behave in a professional manner

careless/reckless behavior increases safety risks for everyone

6

Use proper techniques in handling lab materials and equipment

If you are unsure, ask for instructor's help.

7

Stay out of the stockroom and preparation areas of the laboratory

Areas are off limits to students

8

Know what to do in an emergency

a. report all accidents immediately. All personal injuries -- regardless of how trivial they may seem at the time, must be reported to the instructor and or professional laboratory personnel. College is required by law to file accident reports.

b. Know the procedure to be followed in case of personal injury:
- report it immediately
- in cases of severe injury, notify campus police (3111) and/or dial 911

c. Know what sorts of accidents are most likely to occur in the lab, as well as what action to take if an accident occurs.

9

Wash all glassware, dissecting supplies, etc,. that you use when you are finished with your lab exercise.

Wash all glassware, dissecting supplies, etc,. that you use when you are finished with your lab exercise.

10

Clean your lab table and turn off all hot plates, propane burners, and water faucets. Many students use the lab. No one but you should have to clean up your mess.

Clean your lab table and turn off all hot plates, propane burners, and water faucets. Many students use the lab. No one but you should have to clean up your mess.

11

Never use flammable liquids such as alcohols, gasoline, or ether near a flame.

Read the label carefully before taking any substance from a bottle. Be sure you are using the correct chemical.

12

Only take the amount of chemical necessary to conduct the experiment. Using larger quantities won’t make the experiment work any better. Never return unused chemicals to the reagent bottles.

Only take the amount of chemical necessary to conduct the experiment. Using larger quantities won’t make the experiment work any better. Never return unused chemicals to the reagent bottles.

13

Never throw solid materials (i.e., litmus papers, pH papers, lens paper, toothpicks, animal parts, etc) into the sinks. Dispose of these materials in the appropriate receptacles. Some liquid wastes may be dumped into the lab sinks (check w/ instructor); flush drain with plenty of running water.

Never throw solid materials (i.e., litmus papers, pH papers, lens paper, toothpicks, animal parts, etc) into the sinks. Dispose of these materials in the appropriate receptacles. Some liquid wastes may be dumped into the lab sinks (check w/ instructor); flush drain with plenty of running water.

14

If you have used a microscope during the lab period, clean all microscope lenses with lens paper when you are finished, clean off the microscope stage, shut off the light. Return to its proper shelf.

If you have used a microscope during the lab period, clean all microscope lenses with lens paper when you are finished, clean off the microscope stage, shut off the light. Return to its proper shelf.

15

If you have used prepared microscope slides during the lab period, clean off any immersion oil from the slides, return them to their proper box or tray (check the label).

If you have used prepared microscope slides during the lab period, clean off any immersion oil from the slides, return them to their proper box or tray (check the label).

16

If you are working with live microorgs and spill them on:

a. work surface or floor:
b. skin:
c: clothes

a. work surface or floor: cover w/ paper towel, flood with disinfectant; wait 15 minutes, clean up.
b. Skin: wash area thoroughly with soap and water.
c. Clothes: change clothes; wash and disinfect clothes.

17

When dealing with bodily fluids (saliva, urine, blood), only work with your own, or wear disposable surgical gloves and avoid skin contact.

When dealing with bodily fluids (saliva, urine, blood), only work with your own, or wear disposable surgical gloves and avoid skin contact.

18

Disinfect all work surfaces after working with body fluids or microorganisms. Spray & wipe down tables after every lab.

Disinfect all work surfaces after working with body fluids or microorganisms. Spray & wipe down tables after every lab.

19

Discard all items contaminated with body fluids or microorganisms in designated biohazard containers.

Discard all items contaminated with body fluids or microorganisms in designated biohazard containers.

20

Wash your hands with soap and water before leaving the lab.  

Wash your hands with soap and water before leaving the lab.  

21

FRAMEWORK

arm
base

22

Stage

stage – supports slide
stage adjustment – clamping devise
stage adjustment knob – for holding and moving the slide around on the stage

23

Light source

light intensity control – vary intensity of light, under on/off switch
neutral density filter – sometimes added over the light source in the base, or sometime built into the base

24

Lens system

ocular
objectives
nosepiece
4x
10x
40x
100x
condenser
diaphragm

ocular – eyepiece at the top, has 10x magnification
objectives – (lenses), attached to nosepiece
nosepiece – rotatable, moves objectives around
4x – lowest power, actually 40x (10x * 4x)
10x – “low-power”, actually 100x
40x – “high-dry”, actually 400x
100x – “oil immersion”, actually 1000x
condenser – under stage, collects and directs the light from the lamp to the slide being studied, does not affect magnifying power – moves up and down
Diaphragm – regulates amount light that reaches the slide – moves counter/clockwise

25

Focusing knobs

Coarse adjustment knob
Fine adjustment knob

26

Ocular adjustment

make adjustments on right eye first, then make changes to right eye by turning the diopter adjustment ring

27

Oil immersion

• Rotate oil immersion lens halfway, drop immersion oil, then rotate lens.
• Open diaphragm as high as possible, and keep condenser at highest point.
• Clean after with lens tissue

28

Describe position of hands when carrying microscope to and from your laboratory bench.

Use both hands, one hand on arm, one hand on base.

29

What two adjustments can be made to the condenser? What effect do these adjustments have on the image?

Moves up and down –collects and directs the light from the lamp to the slide being studied

30

Why are condenser adjustments generally preferred over the use of the light intensity control?


Differentiate limit of resolution of typical light microscope vs unaided eye

increases illumination without affecting bulb light


.2mm vs .2um

31

When using the oil immersion lens, what four procedures can be implemented to achieve the maximum resolution?

- oil immersion oil for continuous lens system
- blue filter for shorter wavelengths
- condenser @ highest position
- diaphragm open

32

Why is it advisable to start first with low-power lens when viewing a slide?

Enables you to explore the slide to look for the object you are planning to study

larger working distance prevents you from hitting and breaking the slide.

33

This objective lens provides the highest magnification.

oil immersion

34

This objective lens provides the second highest magnification.

high-dry

35

This objective lens provides the lowest magnification.

low-power

36

This objective lens has the shortest working distance.

100x – oil immersion lens

37

The coarse focus knob should be adjusted only when using this objective lens.

low power or rapid scanning

38

This lens collects and focuses light from the lamp onto the specimen on the slide.




condenser

39

This lens, also known as the eyepiece, often comes in pairs.

ocular

40

Diopter adjustments can be made to this lens.

On the ocular, with the diopter adjustment ring

41

A diaphragm is used to regulate light passing through this lens.

condenser

42

Acetone is the safest solvent for cleaning an objective lens.

False—the best is green soap with warm water or xylene.

43

Only lint-free, optically safe tissue should be used to wipe off microscope lenses.

True

44

The total magnification capability of a light microscope is only limited by the magnifying power of the lens system.

False—ocular lenses provide 10x

45

The coarse focus knob can be used to adjust the focus when using any of the objective lenses.

False—should only be used for lowest power

46

Once focus is achieved at one magnification, a higher-power objective lens can be rotated into position without fear of striking the slide.

True

47

The resolving power of a microscope is a function of:
a. The magnifying power of the lenses
b. The numerical aperture of the lenses
c. The wavelength of the light
d. Both a & b are correct
e. Both b & c are correct

e

48

The coarse and fine focus knobs adjust the distance between
a. The objective and ocular lenses
b. The ocular lenses
c. The ocular lenses and your eyes
d. The stage and the condenser lens
e. The stage and the objective lens

e

49

A microscope that maintains focus when the objective magnification is increased is called
a. Binocular
b. Myopic
c. Parfocal
d. Refractive
e. Resolute

c

50

The total magnification achieved when using a 100x oil immersion lens with 10x binocular eyepieces to
a. 10x
b. 100x
c. 200x
d. 1000x
e. 2000x

d

51

The most useful adjustment for increasing image contrast in low-power magnification is
a. Closing down the diaphragm
b. Closing one eye
c. Opening up the diaphragm
d. Placing a drop of oil on the slide
e. Using a blue filter

a

52

Before the oil immersion lens is rotated into place, you should:
a. Center the object of interest in the preceding lens
b. Lower the stage with use of the coarse focus adjustment knob
c. Place a drop of oil on the slide
d. Both A and C are correct
e. All are correct

d

53

Aseptic Technique

PROCEDURE: Removing organisms from a broth culture with inoculating loop

1. Inoculating loop is heated until it is red-hot.
2. Organisms in culture are dispersed by shaking tube.
3. Tube enclosure is removed and mouth of tube is flamed.
4. Loopful of organisms is removed from tube.
5. Loop is removed from culture and tube mouth is flamed.
6. Tube enclosure is returned to tube.

54

Aseptic Technique

Procedure for inoculating a nutrient broth

1. Cap removed from sterile broth and tube mouth is flamed.
2. Unheated loop is inserted into tube of sterile broth.
3. Loop is removed from broth and tube mouth is flamed.
4. Tube enclosure is returned to tube.
5. Loop is flamed and returned to receptacle.

55

Aseptic Technique

Procedure for inoculating a nutrient agar slant from a slant culture

1. Inoculating loop is heated until it is red-hot.
2. Cap is removed from slant culture and tube moth is heated.
3. Organism is picked up from slant with inoculating loop.
4. Mouth of tube is flamed. Inoculating loop is not flamed.
5. Slant culture recapped and returned to test-tube rack.
6. Tube of sterile agar slant is uncapped and moth is flamed.
7. Slant surface is streaked with unflamed loop in serpentine manner.
8. Tube mouth is flamed, recapped, and incubated.
9. Loop is flamed red-hot and returned to receptacle.

56

Aseptic Technique

Procedure for inoculating a nutrient agar slant from an agar plate

1. Inoculating loop is heated until it is red-hot.
2. With free hand, raise the lid of petri plate just enough to access a colony to pick up a loopful of organisms.
3. After flaming the mouth of a sterile slant, streak its surface.
4. Flame the mouth of the tube and recap the tube.
5. Flame the inoculating loop and return it to receptacle.

57

Aseptic Technique

Provide 3 reasons why the use of aseptic technique is essential when handling microbial cultures in the lab.

• Ensures no contaminating organisms are introduced into culture materials when inoculated or handled in some manner.
• Ensures that organisms that are being handled do not contaminate the handler or others who may be present
• Ensures that no contamination remains after you have worked with cultures

58

Aseptic Technique

Provide two examples of how the Bunsen burner is used during inoculation of a tube culture.

• To sterilize inoculating loop (incinerates any contaminating organisms that may be present)
• To flame the tube

59

Aseptic Technique

How is air contamination prevented when an inoculating loop is used to introduce or take a bacterial sample to/from an agar plate?

Plate cover is raised and held diagonally over the plate to protect the surface from any contamination in the air.

60

Aseptic Technique

Where should a label be written on an agar plate?

On the bottom, on the side with the microorganism.

61

Aseptic Technique

How should agar plates be incubated? Why?

Upside down to prevent moisture from condensing on the agar surface and spreading the inoculated organisms

62

Aseptic Technique

Disinfectant is used on your work surface:
a. Before the beginning of laboratory procedures
b. After all work is complete
c. After any spill of life microorganism
d. Both B and C are correct
e. All of the above are correct.

E

63

Aseptic Technique

To retrieve a sample from a culture tube with an inoculating loop, the cap of the tube is:
a. Removed and held in one’s teeth
b. Removed and held with the fingers of the loop hand
c. Removed with the fingers of the loop hand and placed in the fingers of the tube hand
d. Removed with the fingers of the loop hand and placed on the laboratory bench
e. Any of these methods can be used

B

64

Aseptic Technique

An inoculating loop or needle is sterilized in a flame
a. By one brief passage
b. For exactly 5 minutes
c. Until the entire wire is bright red
d. Until the handle is bright red
e. Until the tip is bright red

C

65

Method A
Pure culture techniques

1. Streak one loopful over area 1 near edge of plate. Apply tightly. Don’t gouge into medium.
2. Flame the loop, cool 5 seconds, make 5-6 streaks from Area 1 through Area 2. Momentary touching the loop to a sterile area of the medium before streaking ensures a cool loop.
3. Flame the loop again, cool, and make 6-7 streaks from Area 2 through Area 3.
4. Flame the loop again, and make as many streaks as possible from Area 3 through Area 4, using up the remainder of the plate surface.
5. Flame the loop before putting it aside.

66

Define colony

identical progeny of the original cell

67

What colony characteristics can be used for differentiation of bacterial species?

Color, shape, other colony characteristics

68

Micrococcus luteus
Color, shape, other colony characteristics

yellow, round, smooth, umbonate, opaque, dull; coccus tetrads, gram positive

69

Serratia marcescens
Color, shape, other colony characteristics

red, round, smooth, convex, opaque, shiny

70

Why is the loop flamed before it is placed in a culture tube? Why is it flamed after completing the inoculation?

Reduce contamination of the microorganism itself; reduce contamination of the tools and for those who have to handle the tools later.

71

Explain why plates should be inverted during incubation.

Prevents moisture from condensing on the agar surface and spreading the inoculated organisms

72

Smear prep from liquid:

1. 2 loopfuls of liquid containing organisms are placed in the center of the target circle
2. Organisms dispersed over entire area of the target circle
3. Smear is allowed to dry to room temp
4. Slide passed through flame several times to heat-kill and fix organisms to slide. Use of clothespin is suggested.

73

Smear prep from solid media:

1. Two loopfuls of water are placed in center of target circle
2. Very small amount of organisms is dispersed with inoculating loop in water over entire area of target circle
3. Smear is allowed to dry to room temp
4. Slide passed through flame several times to heat-kill and fix organisms to slide. Use of clothespin is suggested.

74

Exercise 12: Simple staining with basic dyes

• Methylene blue
• Basic fuchsin
• Crystal violet

All work well because they have color-bearing ions that are positively charged (cationic) –bacteria is negatively charged

75

Gram staining
Gram positive

None (heat fixed) Nothing
Crystal violet Purple
Gram’s iodine Purple
Ethyl Alcohol Purple
Safranin Purple

76

Gram staining- Gram-negative

None (heat fixed) Nothing
Crystal violet Purple
Gram’s iodine Purple
Ethyl Alcohol White
Safranin Pink

77

Crystal violet

primary stain

78

Gram's iodine

complexes w/ crystal violet and forms an insoluble complex with the crystal violet and forms an insoluble complex in gram-positive cells

79

Ethyl alcohol

decolorizes gram-negative bacteria

80

Safranin

counterstains gram-negative bacteria after decolorization

81

Spore staining:

Malachite green & safranin
schaeffer-fulton method
Procedure:
1. Saturate w/ malachite green, steam over boiling water for 5 minutes. Add additional stain if stain boils off.
2. After the slide has cooled sufficiently, remove the paper toweling and rinse with water for 30 seconds.
3. Counterstain with safranin for about 20 seconds.
4. Rinse briefly with water to remove safranin.
5. Blot dry with bibulous paper, and examine slide under oil immersion.

82

acid-fast staining
other name
what is the stain?
sequence

1. Cover smear with carbolfuchsin; steam over boiling water 5 minutes. Add additional stain if stain boils off
2. After slide has cooled, decolorize with acid-alcohol for 15-20 seconds.
3. Stop decolorizing action of acid-alcohol by rinsing briefly with water.
4. Counterstain with methylene blue for 30 seconds.
5. Rinse briefly with water to remove excess methylene blue.
6. Blot dry with bibulous paper. Examine under oil immersion.

aka ziehl-neelson

83

Recognize microscopic slide of acid-fast and non-acid-fast bacteria

Acid fast appears pink or red in stained smears. Most other bacteria are decolorized by the acid-alcohol and are counterstained with methylene blue to be seen.

84

Why is mycobacterium acid fast?

• Has a waxy material in their cell walls (mycolic acid)
• Significantly affects staining properties of these organisms and prevents them from being stained by main of the stains used in microbiology

85

1. Which of the 3 differential stains would likely be the first used when identifying an unknown bacterium?

Gram-stain

86

2. What is the function of a mordant?

complexes with the crystal violet and forms an insoluble complex in gram-positive cells
Causes the primary stain to adhere better or be taken up by the cell so that it is not removed during the decolorizing step

87

3. For differential staining, how does a counterstain differ from a primary stain?

is a different color than the primary stain to aid differentiation

88

4. How do gram-positive and gram-negative bacteria differ in cellular structure? How does this contribute to their differential staining properties?

gram-positive has a thicker cell wall, which retains the crystal violet better in the presence of a decolorizer, compared to gram-negative, which has a thin wall

89

5. Which is the most critical step in Gram-stain procedure? Why? If this procedure is done incorrectly, how might that affect the final results?

Decolorizer step: step in which cells become differentiated.
If too much is used, gram positive cells will lose primary stain and be counterstained pink.
If too little is used, gram negative cells will not lose the primary stain and will remain purple.

90

6. How does culture age affect the results of a gram-stain?

Old cultures of gram positive cells may not retain stain as well as younger cultures, and could give false negative results.

91

7. How does culture age affect the results of a spore stain?

Old cultures of spore formers are ideal because the conditions of nutrient depletion sporulation is more likely to occur.

92

8. Why must smear thickness be considered before performing a gram-stain?

Thick smears will entrap the primary stain, and will not be easily decolorized.

93

9. What color are bacterial endospores after a gram-stain is performed? What does this tell you about the physical property of endospores?

Basic dyes do not penetrate spores so gram staining will result in colorless spores. This indicates that spores are very resistant structures.

94

10. Bacillus anthracis, the causative agent of anthrax, is an endospore former. Why does this trait enhance its capabilities as a biological warfare?

They’re easily produced, can be dispersed in the air, and are environmentally stable.

95

11. What makes Mycobacterium particularly resistant to staining? How are the bacteria in this genus grouped in terms of gram classification?

Has a peptidoglycan layer filled with mycolic acids that make the cell wall waxy and impenetrable to stains. They are classified with gram positive cells because of cell wall thickness and genetic similarities.

96

12. How do you think the acid-fast nature of mycobacterium contributes to its virulence?

Waxy cell wall of mycobacterium protects the bacterium against phagocytosis and some antibiotics while in the host so the pathogen has a greater opportunity to cause disease.

97

1. Color of Staphylococcus aureus before primary stain is added:

no color

gram +

98

2. Color of Pseudomonas aeruginosa after primary stain is added:

purple
gram -

99

3. Color of Bacillus megaterium after mordant is added:

purple
Gram +

100

4. Color of Staphylococcus aureus cells after decolorizer is used:

purple
Gram +

101

5. Color of Moraxella (Branhammella) catarrhalis after decolorizer is used:

no color
gram -

102

6. Color of Bacillus megaterium after counterstain is added:

purple
gram +

103

7. Color of Pseudomonias aeruginosa after the counterstain is added:

pink
gram -

104

Gram stain dyes

primary stain
mordant
decolorizer
counterstain

Crystal violet
Iodine
Alcohol
Safranin

105

Spore stain dyes

primary stain
mordant
decolorizer
counterstain

Malachine green
Heat & water
Rinsed water
Safranin

106

Acid-fast stain dyes

primary stain
mordant
decolorizer
counterstain

Carbolfucsin
Heat
Acid-alcohol mordant
Methylene blue

107

Bacterial cell wall is composed of:

a. Peptidoglycan
b. Phospholipids
c. Proteins
d. Simple polysaccharides
e. Both B and C are correct

a

108

Exosporium (endospore coat) is composed of:
a. Peptidoglycan
b. Phospholipids
c. Proteins
d. Simple polysaccharides
e. Both B and C are correct

c

109

Endospores are produced by bacteria in the genus
a. Bacillus
b. Clostridium
c. Mycobacterium
d. A & B
e. A & C

d

110

Acid-fast staining is useful for identifying the causative agent of:
a. Leprosy
b. Tetanus
c. Tuberculosis
d. A & C
e. All

d

111

Motility

Positive & negative

• Positive: cloudy, diffuse growth (motility)
• Negative: well-defined growth along the stab (no motility)

112

Thioglycolate test—Effects of oxygen on growth

where do the microbes grow?

aerobic
facultative anaerobic

• Aerobic: only at the top
• Facultative anaerobic: all over the tube

113

Obligate (strict) aerobes

MUST grow in oxygen

114

Microaerophiles:

prefer to grow in oxygen concentrations of 2-10% rather than 20% found in atmosphere. Lower concentration is needed for their respiratory metabolism

115

Facultative aerobes (anaerobes):

grow very well aerobically, but also can grow anaerobically if oxygen is not present—metabolism is flexible because under aerobic conditions, they can carry out respiration to produce energy, but if oxygen is absent, they can switch to fermentation that does not require oxygen for energy production

116

Aerotolerant anaerobes:

can tolerate oxygen and even grow in its presence, but do not require oxygen for energy production
• Produces energy strictly by fermentation and not by respiratory means
• Also called Obligate fermenters

117

Obligate (strict) anaerobes:

cannot tolerate oxygen and must be cultured under conditions in which oxygen is completely eliminated. They carry out fermentation or anaerobic fermentation, where inorganic compounds (nitrates, sulfates) replace oxygen for terminal electron acceptor.

118

Is UV light Ionizing or nonionizing? Short/long?

Short, nonionizing. Shorter is more damaging because it has more energy. 260nm is the optimal wavelength of DNA, therefore 260 is most germicidal.

119

Which is more germicidal: short or long wavelength?

Shorter is more damaging because it has more energy. 260nm is the optimal wavelength of DNA, therefore 260 is most germicidal.

120

How does UV light cause death of microbes?

Causes the formation of pyrimidine dimers (formation of covalent bond between neighboring thymine or cytosine molecules in the same DNA strand) that results in the distortion of the double helix DNA structure, which can lead to mutation and death of microbes

121

What limited protection do cells have against the damaging effects of UV radiation?

Photolyases and excision repair mechanism can repair UV damages up to a certain point. Time of exposure is one of the damaging factors. Penetrating ability of UV light also plays a role. Some materials can block UV radiation from reacing the cells.

122

How are endospores protected from the harmful effects of UV light?

They are more resistant to harming effects of UV light than vegetative cells. They are dormant; not replicating. Also, DNA of endospores is protected by small, acid-soluble DNA-binding proteins.

123

What result was observed when the petri dish was kept covered during exposure to UV light? Why was such a result observed?

Unknown, but assumed: paper covered is not damaged.

124

Kirby-bauer method

1. Entire surface of a plate of nutrient medium is swabbed with organism to be tested.
2. Using forceps, take one antibiotic and place it on the inoculated medium.
3. Repeat with all antibiotics in the test.
4. Incubate for 18 hrs.
5. Measure zone of inhibition.
6. Use Kirby-bauer chart to determine whether it is resistant, intermediate, or sensitive.


-- Measure entire diameter --

125

Antibiotic

antimicrobials of usually low molecular weight, produced by microorganisms that inhibit or ill other microorganisms.

126

Semi-synthetic

Antibiotics that are chemically altered to make them more effective in their mode of action

127

Synthetic

antimicrobials that are chemically synthesized in the lab and are not produced by microbial biosynthesis

128

Know how to measure the zone of inhibition, and interpret results using chart when prepared plate is given.

Measuring the area around the disk where no growth occurs.

129

What is the special culture medium that you use for kirby-bauer method?

Mueller-Hinton medium

130

Evaluation of antiseptics: filter paper disk method

1. Inoculate with one loopful of organisms
2. Seeded nutrient agar is poured into plate and allowed to solidify.
3. Sterile disk is dipped halfway into agent.
4. Impregnated disk is placed into center of nutrient agar and pressed down lightly to secure it.
5. After 24-48 hours incubation, the zone of inhibition is measured between disk edge and growth.

131

Fermentation difference

• Difference: see if broth cultures are using lactose or glucose as the sole source of carbohydrate—all can grow lactose, but some will grow aerobically and will ferment lactose with the production of acidic and gaseous by-products of fermentation.

132

Fermentation Medium

• Use Durham tube

o Each tube contains a broth medium with either lactose or glucose as the sole carbohydrate.
o Each tube also contains a pH indicator (phenol red) as well as an inverted vial
o Tube starts out as red (pH medium)
o If acidic products are produced by fermentative growth, the pH will drop, and the indicator will change from red to yellow.
o The inverted vial will trap some fermentatively-produced gases, which may be emitted by the bacteria. If tubes turn yellow or have gas bubbles in the intverted vial, your organism can ferment sugar. (+)

133

Fermentation positive & negative results

o If organism grows but fails to turn the medium yellow or produce gas, it is aerobically metabolizing sugar.

o If tube turns completely yellow and/or has gas bubbles in the inverted vial, organism is capable of fermenting sugar.

o If tube is mostly yellow with a red top, this is due to buildup of carbon-dioxide gas from aerobic growth (-)

134

Fermentation Positive bacterias

o Positive result bacteria: E. coli is facultative anaerobe and is capable of fermenting sugars

135

Fermentation Negative bacteria

o Negative result bacteria: P. aeruginosa is obligate anaerobe, does not cause fermentation of sugars.

136

Kligler’s Test – Hydrogen Sulfide Production principles

• Production of hydrogen sulfide gas – degrades the amino acid cysteine to produce pyruvic acid, ammonia and hydrogen sulfide.
• Iron salts will react with hydrogen sulfide liberated from cysteine to produce insoluble black precipitation of iron sulfide.
• Helpful for differentiation of gram-negative enteric bacteria.
• Tube is streaked first, then stabbed. If agar turns yellow, fermentation has occurred.
• If it stays red, aerobic metabolism is being demonstrated.
• If it turns black, hydrogen sulfide has been produced.

137

Kligler’s Test – Hydrogen Sulfide Production Positive & negative results

• If it stays red, aerobic metabolism is being demonstrated.
• If it turns black, hydrogen sulfide has been produced.

138

Kligler’s Test – Hydrogen Sulfide Production Positive bacteria

Proteus vulgaris, Salmonella typhimurium

139

Kligler’s Test – Hydrogen Sulfide Production Negative bacteria

E. coli, P. aeruginosa

140

Citrate Test principles

• Can use citrate as a sole carbon source during oxidative metabolism
• Culture medium used is Simmon’s Citrate Agar, also contains ammonium salts that serve as a nitrogen source for growth.
• Bacteria that cleave citrate must also use ammonium salts, and in the process, they produce ammonia that causes the medium to become alkaline.

141

Citrate Test positive & negative results

• If alkaline, pH indicator in the dark green changes to dark blue color.

142

Citrate Test positive bacteria

E. cloaca, S. typhimurium

143

Citrate Test negative bacteria

E. coli

144

Isolation of gram negative intestinal bacteria using EMB agar principles

• EMB =Eosin-Methylene Blue – contains lactose and a dye so that if an organism is a lactose fermenter, its colony will take on a color characteristic of the dye present.

145

E. coli EMB agar results

produces brilliant, metallic green isolated colonies with black centers

146

Enterobacter cloacae EMB agar results

produces mucoid, pinkish spreading colonies

147

Salmonella typhimurium EMB agar results




produces translucent, colorless-to-amber colonies

148

Proteus vulgaris EMB agar results

produces spreading colorless colonies

149

Pseudomonas aeruginosa EMB agar results

produces clear colonies with dark centers

150

Identification of certain gram-positive bacteria using mannitol salt agar principles

• Mannitol salt agar is a selective medium for Gram-positive bacteria that can tolerate salt.
• Starts pink—if it ferments, it becomes yellow; if it does not grow, it stays pink
• Tests toleration to salt
• Differential medium

151

Mannitol salt agar positive bacteria

staphylococcus

152

Mannitol salt agar negative bacteria

Streptococci

153

Mannitol salt agar positive and negative results

Ferments mannitol with the production of acids, turns the medium completely yellow

154

Gelatin stab test principles

• Motility test—use inoculation needle

155

Gelatin stab test positive and negative results

• If it liquefies, it has gelatinase which can break down the gelatin
• If it does not liquefy, it does not have gelantinase and it does not break down gelatin.

156

Gelatin stab test positive bacteria

B. subtilis

157

Gelatin stab test negative bacteria

E. coli

158

Urease principles

• Yellow, hint of orange; becomes red/pink (produces urease)
• They can break down urea into ammonia and CO2

159

Urease positive and negative results

• Increase of pH changes the phenol red in the medium from yellow to a bright pink color

160

Urease positive bacteria

Proteus vulgaris

161

Urease negative bacteria

E. coli

162

Catalase principles

• When aerobic bacteria grow by aerobic respiration, they use oxygen as a terminal electron acceptor, converting it to water. They can also produce hydrogen peroxide as a by-product, which is a highly reactive oxidizing agent that can damage enzymes, nucleic acids, and other bacterial cell components.
• To avoid this damage, aerobic bacteria produce the enzyme catalase, which degrades hydrogen peroxide to harmless oxygen and water. Anaerobes and aerotolerant bacteria lack this enzyme.
• Differentiates between aerobic and anaerobic

163

Catalase positive and negative results

• If peroxide bubbles at all, organisms produces the enzyme catalase, which catalyzes the conversion of hydrogen peroxide to water and oxygen gas (bubbles)

164

Catalase positive bacteria

Staphylococcus

165

Catalase negative bacteria

Streptococcus

166

Beta-hemolysis

Complete lysis of red blood cells around a colony and results in a clear zone surrounding the colonies

167

Alpha-hemolysis

partial break down, producing a greenish discoloration around the colonies

168

Gamma-hemolysis

do not exhibit any hemolysis of blood display, no effect on red blood cells in a blood agar plate.