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Clinical Bacteriology > (P) Lec 4: Culture Staining (Part 1) > Flashcards

(P) Lec 4: Culture Staining (Part 1) Flashcards

1
Q

Flow of Lab Procedures for the Diagnosis of Infectious Diseases

This is the first step upon receiving the specimen

A

Direct Examination by Microscopy

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2
Q

Flow of Lab Procedures for the Diagnosis of Infectious Diseases

What sample is not directly examined in microbiology due to the abundance of gram-negative bacteria present in it?

A

Feces

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3
Q

Flow of Lab Procedures for the Diagnosis of Infectious Diseases

Feces predominantly contains this type of bacteria hence why direct examination via microscopy is not advised

A

Gram (-) bacilli

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4
Q

Flow of Lab Procedures for the Diagnosis of Infectious Diseases

This kind of bacteria in feces can still cause dysentery despite not performing gram staining on it

A

Gram (+) bacteria

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5
Q

Flow of Lab Procedures for the Diagnosis of Infectious Diseases

Gram (+) bacteria in stool can cause what illness?

A

Dysentery (infection in your intestines that causes bloody diarrhea)

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6
Q

Flow of Lab Procedures for the Diagnosis of Infectious Diseases

This is when you inoculate bacteria in an enrichment broth or plated agar to save the specimen

A

Culture

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7
Q

Flow of Lab Procedures for the Diagnosis of Infectious Diseases

This is agar used in culturing which is placed on a petri dish

A

Plated agar

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8
Q

Flow of Lab Procedures for the Diagnosis of Infectious Diseases

This specimen is usually plated on BAP, MAC, CAP, and Theyer-Martin

A

Urethral discharge

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9
Q

Flow of Lab Procedures for the Diagnosis of Infectious Diseases

Urethral discharge is put into a tube containing what broth?

A

Thioglycollate

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10
Q

Flow of Lab Procedures for the Diagnosis of Infectious Diseases

How many hours must cultures be isolated for?

A

16 to 24

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11
Q

Flow of Lab Procedures for the Diagnosis of Infectious Diseases

Pure cultures (which contain the pathogenic agent) are usually found where on the plate?

A

Last part where you swabbed

First area swabbed = concentrated bacteria (a mix of everything)

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12
Q

Flow of Lab Procedures for the Diagnosis of Infectious Diseases

This step is done after culturing your specimen

A

Analysis of Cultivated Organisms (identification and susceptibility testing)

e.g. biochemical testing for specific pathogens

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13
Q

Flow of Lab Procedures for the Diagnosis of Infectious Diseases

Biochemical testing and susceptibility testing must be done in what manner?

A

Simultaneously (at the same time)

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14
Q

This refers to the use of a microscope to magnify objects too small to be visualized with the naked eye so that their characteristics are readily observable

A

Microscopy

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15
Q

Applications of Microscopy

Fill in the blanks:
1. For (blank) identification of organisms (e.g. is it gram [+] or [-]?)
2. Rapid final identification by direct (blank) commonly used for parasites
3. Detection of (blank) organisms in the same specimen
4. Detection of organisms not easily (blank) in the laboratory

A
  1. Preliminary
  2. Visualization
  3. Different
  4. Cultivated
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16
Q

Applications of Microscopy

Fill in the blanks:
1. Evaluation of patient specimens for the presence of cells indicative of (blank) or contamination
2. Provide (blank) information about which organisms are expected to (blank) so that appropriate techniques are used
3. Determine which tests and methods should be used for identification and (blank) of cultivated organisms

A
  1. Inflammation
  2. Preculture & Grow
  3. Characterization
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17
Q

Applications of Microscopy

What bacterial species stated in the video is known to be hard to cultivate/grow in a culture due to it being a slow grower?

A

Mycobacterium tuberculosis

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18
Q

Applications of Microscopy

Mycobacterium tuberculosis are slow growers which need how long to grow?

A

Weeks

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19
Q

Applications of Microscopy

Sputum samples should usually have more than how many PMNs and less than how many epithelial cells per low-power field?

A

PMNs = 25
Epithelial Cells = 10

If values deviate, reject it under the impression that it is not sputum

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20
Q

Applications of Microscopy

What is the gold standard in identifying bacterial species?

A

Culture and sensitivity

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21
Q

Applications of Microscopy

The identity of the bacteria found must be aligned with its what?

A

Workups that are to be done (set of tests)

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22
Q

Microscopes

Principle: Visible light is passed through the specimen and then through a series of lenses that bend the light in a manner that results in magnification of the organisms present in the specimen

A

Bright Field Microscope (normal microscope)

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23
Q

Microscopes

How do you compute for total lens magnification?

A

Objective lenses used multiplied by the ocular lens (40X, 100X, 400X, and 1000X)

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24
Q

Microscopes

This is the property of the lens to completely separate two objects in a microscopic field

A

Resolving Power

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25
Q

Microscopes

To maximize the resolving power of the brightfield microscope (fill in the blanks):
1. Use (blank) filter placed over the light source because a shorter (blank) will provide maximum resolution
2. The (blank) must be kept at the highest position
3. The (blank) should not be stopped down too much
4. The use of (blank) oil

A
  1. Blue & Wavelength
  2. Condenser
  3. Diaphragm
  4. Immersion
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26
Q

Microscopes

This is used in staining techniques

A

Contrast

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27
Q

Microscopes

We stain bacteria because a percentage of its cellular content is water, making the contrast minimal. What percentage of it is water?

A

80%

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28
Q

Microscopes

Principle: Utilizes beams of light passing through the specimen that are partially deflected by the different densities or thicknesses of the microbial cells or cell structures in the specimen

A

Phase Contrast Microscope

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29
Q

Microscopes

TOF: In Phase Contrast Microscope, staining is needed in order to view the viable forms of the bacteria

A

False (staining is not needed)

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30
Q

Microscopes

This kind of microscope offers the advantage of allowing for the observation of viable organisms

A

Phase Contrast

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31
Q

Microscopes

Principle: Uses fluors (or fluorochrome) to raise it to a higher energy level after absorbing UV light. When the dye molecules return to their normal, lower energy state, they release excess energy in the form of light

A

Fluorescent Microscopy

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32
Q

Microscopes

TOF: Fluorochroming utilizes antigen-antibody complexes to produce a result that is specific for the target antigen

A

False (immunofluorescence; fluorochroming will stain and light up all bacteria present)

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33
Q

Microscopes

The color of the fluorescent light depends on the usage of what? (2 answers)

A

Dye and light filter used

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34
Q

Microscopes

What are the 3 common dyes used in fluorescent microscopy?

A
  1. Acridine Orange
  2. Auramine
  3. Fluorescein isothiocyanate (FITC)
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35
Q

Microscopes

In fluorescent microscopy, acridine orange, auramine, and FITC require what color of excitation light?

A

Blue

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36
Q

Microscopes

In fluorescent microscopy, calcofluor white requires what color of excitation light?

A

Violet

Commonly used on fungi

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37
Q

Microscopes

Principle: The condenser does not allow light to pass directly through the specimen but directs the light to hit the specimen at an oblique angle. Only light that hits objects will be deflected upward into the objective lens for visualization.

A

Dark Field Microscopy

38
Q

Microscopes

Dark field microscopy is commonly used for what type of bacteria?

A

Spirochetes (are very thin)

39
Q

Microscopes

In dark field microscopy, all other light that passes through the specimen will miss the objective, thus making the background what?

A

A dark field

40
Q

Microscopes

TOF: Staining is not required in dark field microscopy

A

True

41
Q

Microscopes

Principle: Uses electrons instead of light to visualize small objects and, instead of lenses, the electrons are focused by electromagnetic fields to form an image on a fluorescent screen

A

Electron Microscopy

42
Q

Microscopes

Electron microscopy is usually used for what organisms?

A

Viruses (as they are very small)

43
Q

Microscopes

What is the magnification of the electron microscope?

A

100,000X (girl parang makikita mo na kaluluwa ng bacteria)

44
Q

Types of Electron Microscopes

This passes the electron beam through objects and allows visualization of its internal structure

A

Transmission Electron Microscope (TEM)

Clue: In(t)ernal structures = (T)ransmission Electron Microscope

45
Q

Types of Electron Microscopes

This uses electron beams to scan the surface of objects and provides three dimensional views of surface structure

A

Scanning Electron Microscope (SEM)

Clue: (S)urface structures = (S)canning electron microscope

46
Q

Staining Techniques

Bacterial motility cannot be demonstrated with a regular smear as the cover slip will prevent it from showing, it must be prepared through what method?

A

Hanging drop method

47
Q

Staining Techniques

This method requires putting vaseline or petroleum jelly on the corners of the cover slip to prevent sinking

A

Hanging drop method

48
Q

Staining Techniques

TOF: In hanging drop method, you put the sample on the glass slide before putting on the cover slip

A

False (put the sample on the cover slip)

49
Q

Staining Techniques

This technique uses a single stain to color a bacterial cell

A

Simple Staining

50
Q

Staining Techniques

Simple staining only uses what kind of dyes due to them having color-bearing ionic groups (chromophores) that are positively charged (cationic)

A

Basic

The stain is (+) charged while bacteria are (-) charged

51
Q

Staining Techniques

Staining can determine what characteristic of the bacteria?

Clue: cocci, bacilli, etc.

A

Morphology

52
Q

Staining Techniques

If you stain bacteria (negatively charged) with an acid stain (also negatively charged), what will you stain?

A

The background

53
Q

Staining Techniques

Simple stains are informative with what bacteria based on the ff. characteristics:
- Pleomorphism: irregularity of form
- Metachromatic granules: reddish purple
- Palisading arrangement

Identify the bacteria, basically

A

Corynebacterium

54
Q

Staining Techniques

What does the pleomorphism of Corynebacterium mean?

A

Irregular shapes

55
Q

Staining Techniques

What color are the metachromatic granules of Corynebacterium?

A

Reddish-purple

56
Q

Staining Techniques

This is an organelle considered to be the storage form of food or energy for the bacteria (has ionic phosphates)

A

Volutin/Metachromatic Granules

57
Q

Staining Techniques

What is the arrangement of Corynebacterium?

A

Palisading/Picket-fence

58
Q

Staining Techniques

What are the 3 simple stains shown in the powerpoint?

A
  1. Methylene blue
  2. Basic fuchsin
  3. Crystal violet
59
Q

Staining Techniques

This is useful in demonstrating the morphology of bacterial cells & characterizing some of their external structures. It will not stain the organism, only the background which results in indirect staining.

A

Negative Staining

60
Q

Staining Techniques

What kind of dyes are used in negative staining?

A

Acidic (India Ink and Nigrosin are negatively charged which is repelled by the cell wall)

61
Q

Staining Techniques

This is not a negative stain since crystal violet (the reagent) will stain the organism (e.g. Anthony stain)

A

Capsular Staining

62
Q

Staining Techniques

Match the following for capsular staining:
1. Reagent
2. Decolorizer
3. Counterstain

A. Crystal violet
B. 20% copper sulfate

A
  1. A
  2. B
  3. B
63
Q

Staining Techniques

In capsular staining, what color does the capsule stain as?

A

Light blue (OR a white halo)

64
Q

Staining Techniques

Gram staining was developed by who when he wanted to differentiate bacterial cells from eukaryotic nuclei in diseased lungs?

A

Hans Christian Gram

65
Q

Staining Techniques

This is often the first test conducted for unknown samples as it can provide a presumptive identification of the organism

A

Gram Staining

66
Q

Staining Techniques

Culture and Sensitivity will produce results in how many days?

A

3-5 days

67
Q

Staining Techniques

Gram staining differentiates cells based on what 2 components of its structure?

A

Cell wall and composition

68
Q

Staining Techniques

When staining, you usually perform this step first

A

Heat fix or air-dry

69
Q

Staining Techniques

TOF: All stains can be heat fixed

A

False (e.g. endospore stains and capsular stains cannot as it could destroy the capsule)

These only need to be air dried

70
Q

Staining Techniques

Iodine (mordant) when combined with crystal violet forms what in Gram-Positive bacteria?

A

An insoluble complex

71
Q

Staining Techniques

TOF: Acetone is also a decolorizer

A

True

72
Q

Staining Techniques

Gram (+) or Gram (-)?
Thick peptidoglycan layer (teichoic acids)

A

Gram (+)

73
Q

Staining Techniques

Gram (+) or Gram (-)?
An outer membrane covering a thinner peptidoglycan layer

A

Gram (-)

74
Q

Staining Techniques

The crystal violet stain will be retained in some bacteria, while others will not and will appear red from the counterstain (mixing of stains). What kind of organisms manifest this?

A

Gram Variable Bacteria

75
Q

Staining Techniques

TOF: Bacteria that have slimy cell walls are considered gram variable bacteria

A

False (waxy, not slimy)

e.g. Acid-Fast Organisms like Nocardia and Mycobacterium

76
Q

Factors to Consider in Gram Staining

Fill in the blanks:
1. Old culture; more than (blank) hours
2. Must prepare (blank) smears
3. (Blank) is the most critical step

A
  1. 18 hours
  2. Thin
  3. Decolorization
77
Q

Staining Techniques

TOF: Old cultures can turn gram negative to gram positive or variable

A

False (gram positive to negative or variable)

78
Q

Staining Techniques

TOF: Underapplication of the decolorizer (alcohol) may cause the dye-mordant complex to be removed from gram-positive cells causing them to appear gram-negative

A

False (over application)

79
Q

Staining Techniques

Used for bacillus and clostridium

Clue: Think about what classification these 2 belong in

A

Spore Staining

80
Q

Staining Techniques

These 2 bacteria form endospores

A

Bacillus and Clostridium

81
Q

Staining Techniques

These are dehydrated, not metabolically active (dormant), resistant to heat, radiation, acids, and chemicals (disinfectants), and not easily penetrated with stains (needs to be heated)

A

Endospores

82
Q

Staining Techniques

When do Bacillus and Clostridium form endospores (dormant form)?

A

When their energy is depleted

83
Q

Staining Techniques

When sporeformes attain energy again, from an endospore they will turn into what?

A

Their vegetative states

84
Q

Types of Spore Stains

Uses malachite green to stain the endospore, heat is the mordant, water is the decolorizer, and the counterstain is safranin for the vegetative portion

A

Schaeffer-Fulton Method

Stains the endospore green

85
Q

Types of Spore Stains

Produces red spores within colorless sporangium; uses carbol fuchsin and nigrosin (a negative stain which stains the background)

A

Dorner Method

86
Q

Staining Techniques

This is designed to stain bacterial cell walls that contain long-chain fatty (mycolic) acids (or hydromythoxin acid)

A

Acid-Fast Staining

87
Q

Staining Techniques

The mycolic acid of AFB are resistant to what?

A

Decolorization

88
Q

Staining Techniques

What are the 3 AFBs?

A
  1. Mycobacterium spp.
  2. Nocardia spp.
  3. Cryptosporidium spp.
89
Q

2 Methods of AFS

Determine the method:
> Primary Stain: Carbol Fuchsin
> Mordant: Phenol (high concentration)
> Decolorizer: Acid Alcohol (3% HCl in 95% ethanol)
> Counterstain: Methylene Blue

A

Cold Method/Kinyoun Acid Fast

90
Q

2 Methods of AFS

Determine the method:
> Primary Stain: Carbol Fuchsin
> Mordant: Heat/Phenol (low concentration)
> Decolorizer: Acid Alcohol (3% HCl in 95% ethanol)
> Counterstain: Methylene Blue

A

Hot Method/Ziehl Neelsen Acid Fast Method

91
Q

Staining Techniques

Carbolfuchsin contains what?

A

Phenol (an alcohol)

92
Q

Staining Techniques

This is a corrosive combustible poison and should be handled carefully

A

Phenol (an alcohol)

Clinical Bacteriology (9 decks)

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