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BACTERIOLOGY > Methods for examination > Flashcards

Methods for examination Flashcards

Role of microscopy in microbiology

1
Q

Three important steps in preparing bacteriologic samples

A

Step 1: Preparation of smear
Step 2: Fixation
Step 3: Staining

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2
Q

Bacterial smear is properly processed if

A
  • Bacteria are evenly spread out
  • Bacteria are not washed off during staining
  • Bacterial form is not distorted
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3
Q

It is the process wherein the internal and external structures of the cells/microorganism are attached jn a position to the slide. Without this step, the stain might wash the organism off the slide.

A

Fixation

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4
Q

Fixation is achieved either by:

A
  • Heating or application of chemical agents
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5
Q

Chemical agents for fixation includes:

A
  • Ethanol, acetic acid, mercuric chloride

- Formaldehyde and glutaraldehyde

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6
Q

These are salts composed of positive and negative ions, one of which is colored and is known as the chromophore

A

Stains

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7
Q

For basic dyes the color is on what ion?

A

positive ion

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8
Q

For acidic dyes the color is on what ion?

A

negative ion

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9
Q

Why is bacteria more attracted to basic dyes?

A

Bacteria are slightly negatively charged at pH 7 and a basic dye is a stain that is cationic (positively charged) and will therefore react with material that is negatively charged.

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10
Q

This is a staining technique in which single staining agent is used and the main purpose is to highlight the entire microorganism so that shape and some basic structures are visible.

A

Simple staining

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11
Q

A staining technique that divides bacteria into separate groups based on their staining properties .

A

Differential staining

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12
Q

A staining technique in which some specific parts of thr microorganism are colored such as the endospores, flagella, and capsule.

Examples are:
Negative staining for capsules
Schaeffer-Fulton for endospore staining
Leifson method for flagella

A

Special Staining

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13
Q

Two most common differential staining methods in the clinical microbiology laboratory

A
  • Gram staining and Acid-fast staining
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14
Q

Color of gram positive bacteria

A

Blue to purple

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15
Q

Color of gram negative bacteria

A

Pink

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16
Q

This type of staining is used to stain bacteria that have a high lipid and wax content in their cell walls and do not stain well with traditional bacterial stains.

A

Acid-fast staining

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17
Q

The most commonly isolated acid-fast bacteria in the diagnostic microbiology lab

A

Mycobacteria

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18
Q

Acid-fast bacteria will stain _____ in smear while non-acid fast bacteria will appear ____?

A

Red - acid fast bacteria

Blue- not acid fast bacteria

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19
Q

Why do acid fast bacteria stain red?

A

Acid-fast bacteria retain carbol fuchsin so they appear red.

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20
Q

Why do gram negative bacteria appear pink-red after alcohol-acetone decolorization?

A

Gram negative bacteria have thinner cell walls compare to gram positive bacteria. The decolorizer in gram staining procedure damages these thin lipid walls and allows the stain complex to wash out, thus when they are counterstained with red safranin dye they appear pink- red.

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21
Q

Process of gram staining

A

Fixation - specimen is colorless
↓
Crystal violet - specimen turns into deep shade of blue
↓
Iodine treatment - act as mordant
↓
Decolorization
↓→ remains blue - gram positive
Turns transparent - gram negative
↓
Counterstain with safranin dye

result: Gram positive - blue to purple
Gram negative - pink to red

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22
Q

Gram negative bacteria that stain weakly in gram staining

A

Borella, Legionella, and Spirillium spp

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23
Q

Gram negative bacteria that have strong avidity with safranin dye and stain bright red

A

Enterics

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24
Q

A special staining technique in which the background is darkly stained by nigrosin or indian ink, leaving the specific part untouched and thus is visible.

clue: this part is resistant to stain and are only made visible by negative staining technique

A

Capsule staining

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25
Q

Staining method for flagella

A
  • Leifson method

- Gray method (basic fuchsin)

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26
Q

This is the most commonly used endospore staining method in the lab

A

Schaeffer- Fulton

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27
Q

A primary stain used to stain endospores

A
  • Malachite green

Note: since the endospore resist staining the malachite green will be forced into the endospore by heating

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28
Q

The slide is evaluated for the:

A
  • Presence of bacterial cell
  • Gram reactions (positive or negative)
  • Morphologies (cocci or bacilli)
  • Arrangement (chains, pairs, clusters)
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29
Q

Cytocentrifugation is an excellent method for which of the following type of samples?

a. heavily contaminated
b. nonviscous fluids
c. thick purulent material
d. filled with mucous

A

B. nonviscous fluids

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30
Q

In a Wright Giemsa-stained smear, bacteria would appear as which of the following colors?

a. red
b. blue
c. purple
d. orange

A

B. blue

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31
Q

True or False: It is acceptable to prepare smears from swabs after they have been used to inoculate culture media.

A

False

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32
Q

Which of the following stains may be used for direct smear examination?

A.) Gram Stain
B.) Acid-fast Stain
C.) Wright's or Giemsa Stain
D.) Calcofluor White Stain
E.) All of the above
A

E. All of the above

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33
Q

Which of the following stains is best used to detect MYCOBACTERIAL organisms in clinical samples?

A.) Gram Stain
B.) Giemsa Stain
C.) Acid-fast Stain
D.) Lacto-phenol-cotton blue stain

A

C. Acid fast stain

Note: mycobacteria is the most commonly isolated acid-fast bacteria in lab

34
Q

Calcofluor white is a colorless dye that binds with which of the following structures?

A.) Cell wall containing mycolic acid
B.) Chitin
C.) Peptidoglycan layer
D.) Metachromatic Granules

A

B. Chitin

35
Q

A negative stain results in ________ staining of the cell

A

Indirect

  • The background surrounding of the cell is colored by a negative stain.
  • Usually cells appear as transparent objects against a dark background.
36
Q

What are some examples of negative stains?

A

India ink and nigrosin

  • These stains are considered acidic dyes because they have anionic chromophores.
  • Used for capsule staining
37
Q

How many methods are there for negative staining

A

Three

38
Q

Simple staining

A

The use of a single stain to color a bacterial cell.

39
Q

What are some of the most commonly used dyes for simple staining? What are these dyes classified as?

A
  • Methylene blue
  • Basic fuchsin
  • Crystal violet

-Basic dyes (dyes that have positive chromophores)

40
Q

Procedure for simple staining:

A
  1. A bacterial smear is stained with a basic dye for one minute.
  2. The stain is then gently rinsed off with deionized water.
  3. Water drops are carefully blotted off the slide with bibulous paper.
  4. The slide is then examined under oil immersion.
41
Q

What do simple stains allow use to observe?

A
  • Basic morphology

- The presence of absence of certain kinds of granules.

42
Q

Does the first step in preparing a bacteriological smear differ according to the source of the organism?

A

Yes

  • If the bacteria are growing in a liquid medium (broth, milk, saliva, urine, etc.) one starts by placing two or more loopfuls of the liquid medium directly on the slide.
  • From solid media (nutrient agar, blood agar, or some part of the body) one starts by placing one or two loopfuls of water on the slide and then using an inoculating loop to disperse the organisms in the water.
43
Q

What is important to remember when using solid media?

A

Bacteria growing on solid media tend to cling to each other and must be dispersed sufficiently by dilution in water; unless this is done, the smear will be too thick.

44
Q

How much bacteria should be used for making slide from solid media?

A

Only a very small amount is necessary to make a good smear.

45
Q

What kind of stain is Congo Red?

A
  • Acidic (negative chromophores), repelled by bacterial cell.
  • pH sensitive: WIll turn blue at low pH.
  • Will cause the background of the slide to turn purple.
46
Q

What kind of stain is Manevals?

A
  • Basic
  • Contains acid fuchsin
  • Will stain the bacteria cell and turn it a pinkish-red
47
Q

Who discovered the gram stain and when?

A

1884, Danish Physician Christian Gram.

  • He was trying to develop a staining procedure that would differentiate bacterial cells from eukaryotic nuclei in stained tissue samples.
  • He was not successful in developing a tissue stain but what resulted is the most important stain in bacteriology.
48
Q

The gram stain is an example of a _______ stain

A

Differential
-These staining reactions take advantage of the fact that cells or structures within cell display dissimilar staining reactions that can be distinguished by the use of different dyes.

49
Q

Why is gram-positive purple?

A

The first stain that is applied in crystal violet, which stains both gram-negative and gram-positive bacteria purple.

  • Gram’s Iodine is then applied, both gram-negative and gram-positive bacteria remain purple.
  • When Ethyl Alcohol is applied to the bacteria, the gram-positive bacteria becomes dehydrated and closes the pores in its cell wall. Gram-positive becomes dehydrates because of the thick layer of peptidoglycan in its cell wall. Once it becomes dehydrates the crystal violet-grams’ iodine complex remains in the cell. Gram-negative bacteria does not become dehydrated and its pore in the cell wall stay open because it has a very thin layer of peptidoglycan. Once ethyl alcohol is applied to the gram-negative bacteria, the crystal violet-gram’s iodine complex is extracted (decolorization occurs). Leaving the gram-negative bacteria colorless. Therefore, gram-negative bacteria must be counterstained with a red dye; safranin.
50
Q

What is the primary stain used in gram staining?

A

Crystal violet

51
Q

What is Gram’s Iodine?

A

A mordant.

-It complexes with the Crystal Violet and forms an insoluble complex in gram-positive cells.

52
Q

Ethyl Alcohol

A

Important in the decolorization process.

-Ethyl alcohol will remove the crystal violet-iodine complex from gram-negative bacteria but not gram-positive bacteria

53
Q

Safranin

A

Counterstain

  • Once the crystal violet-iodine complex is removed from gram-negative bacteria, the bacteria is colorless.
  • The safranin then stains the gram-negative bacteria, this results in the gram-negative bacteria being pink in color.
  • The safranin does nothing to the gram-positive cells because crystal-violet is a much more intense stain than safranin and the cell wall of gram-positive bacteria is dehydrated and the pores are closed, meaning that the safranin cannot get into gram-positive cells.
54
Q

What factors can affect the outcome of the gram staining procedure?

A
  1. It is important to use cultures that are 16-18 hours old. Gram-positive cultures older than this can convert gram-variable or gram-negative and give erroneous results. (It is important to note that gram-negative bacteria never convert to gram-positive)
  2. Prepare thin slides. Thin smears allow the observation of individual cells and any arrangement in which the cells occur. The thickness of your smears can affect decolorization. Thick smears can entrap the primary stain, which is not removed by alcohol or acetone. Cells that occur in the entrapped stain can appear gram-positive leading to erroneous results.
  3. Decolorization is the most critical step in the gram-stain procedure. If the destaining agent is over applied, the dye-mordant complex can eventually be removed from gram-positive cells, converting them to gram-negative cells.
55
Q

Procedure for Gram-Staining

A
  1. Cover the smear with Crystal Violet and let stand for 1 minute
  2. Briefly wash off the stain, using a wash bottle of distilled or deionized water. Drain off excess water.
  3. Cover the smear with Gram’s Iodine solution and let stand for 1 minute. Gram’s Iodine is a mordant and will make the bacteria larger.
    4, Wash off the Gram’s Iodine. Hold the slide at a 45-degree angle and allow the 95% alcohol to flow down the surface of the slide. Do this until the alcohol is colorless as it flows from the smear down the surface of the slide. This should take no more than 20 seconds for properly prepared smears.
  4. Stop decolorization by washing the slide with a gentle stream of water.
  5. Cover the smear with safranin for 1 minute
  6. Wash gently for a few seconds, blot dry with bibulous paper and air-dry
  7. Examine the slide under oil immersion.
56
Q

What species of bacteria produce spores?

A

Bacillus and Clostridia

57
Q

What are characteristics of endospores?

A
  • Dehydrated
  • Not actively metabolizing
  • Resistant to heat, radiation, acids, and many chemicals
58
Q

Heat resistance of the spore is associated with what?

A
  • Water content

- The higher the water content of the endospore, the less heat resistant the endospore will be.

59
Q

How to destroy endospores by heating:

A

They must be exposed for 15-20 minutes to steam under pressure, which generates temperatures of 121 degrees Celsius,
-Such conditions are produced in autoclaves.

60
Q

What must be applied during the staining process of endospores?

A

Heat

  • If heat is applied while staining with malachite green, the stain penetrates the endospore and becomes entrapped in the endospore.
  • The malachite green is not removed by subsequent washing decolorizing agents or water.
  • In this instance, heat is acting as a mordant to facilitate the uptake of the stain.
61
Q

What stains are used during spore staining?

A
  • Malachite green will stain the endospore
  • Safranin will stain the vegetative cell portion.

Note: green ang endospore and pink-red si vegetative cells

—— This is differential staining—-

62
Q

To facilitate the staining of acid-fast bacteria:

A

It is important to use methods that make the cells more permeable to stains because the mycolic acid is their cell walls prevents the penetration of most stains.

63
Q

What method is used during the acid-fast staining?

A

Kinyoun acid-fast method

  • A modification of the Ziehl-Neelson method. The primary stain is basic fuchsin instead of carbolfuchsin. Phenol is used in both methods but unlike the Ziehl-Neelson method, the Kinyoun acid-fast method does not heat require heat during the procedure.
  • In the Kinyoun acid-fast method the increased concentrations of basic fuchsin and phenol are sufficient to allow the penetration of the stain into cells and the basic fuchsin is not removed during destaining with acid-alcohol.
  • Since heating is not required in this method, phenol fumes are not generated during staining of the cells
64
Q

In the acid-fast staining method, acid-fast bacteria will be what color? What color will non-acid fast bacteria be?

A
  • Acid-fast bacteria will be pink in color (Mycobacterium)

- Non-acid fast bacteria will be blue. (Staphylococcus)

65
Q

Acid-Fast Staining Procedure

A
  1. Prepare a mixed culture smear by placing tow loopfuls of S. aureus on a slide and transferring a small amount of M. smegmatis to the broth on the slide with an inoculating loop. Since the Mycobacteria are waxy and tend to cling to each other in clump, break up the masses of organisms with the inoculating needle. After air-drying the smear, heat-fix it.
  2. Cover the heat fixed smear with basic fuchsin and stain for 5 minutes
  3. Wash with water and shake off any excess water
  4. Decolorize with acid-alcohol for approximately 1 minute. Check by tilting the slide and adding more destain to make sure than no more stain runs off of the smear.
  5. Stop decolorization by the acid-alcohol by rinsing briefly with water.
  6. Counterstain with methylene blue for 1 minute
  7. Rinse briefly with water to remove excess methylene blue.
  8. Blot dry with bibulous paper. Examine under oil immersion.
66
Q

Kinyoun acid-fast method

A

Is a modification of the Ziehl-Neelsen method in which the concentrations of the primary stain, basic fuchsin and phenol, are increased but the bacterial cells are not heated during the staining procedure.

67
Q

Before heat fixing, the slide should be _______

A

Completely air-dried

link: https://quizlet.com/26730093/microbiology-lab-test-set-2-flash-cards/

68
Q

The microscope routinely used for observing microbial cells in the laboratory

A

Coumpound microscope

  • gives wider field of view, less distortion, and less eye strain
69
Q

Part of the microscope that forms a real image inside the microscope tube

Clue: closer to the specimen

A

Objective lens

-usually 10x, 40x, 100x magnification

70
Q

Part of microscope that together with the eye they form a virtual image

Clue: closer to the eye

A

Ocular lens

-10x magnification

71
Q

It used to differentiate microorganism from background materials and debris

A

-Contrast

72
Q

Two major bacterial groups gram staining would differentiate

A

Gram positive and negative bacteria

73
Q

When viewing under the microscope, what does the oil immersion provide or do?

A

gives higher refractive index

74
Q

This type of microscope is used when examining live micro-organisms that are invisible in ordinary light microscope, cannot be stained (unstained) by standard methods, or specimens that are so distorted by staining

A

Darkfield microscopy

-instead of normal condenser, darkfield condenser is used which contain an opaque disc

75
Q

Type of Electron microscope that creates 3D images ___________
2D images _____________

A

SEM- 3D

TEM -2D

76
Q

Bacteria that takes up the primary basic dye (crystal violet) used in gram staining

A

-Gram positive bacteria

77
Q

Bacteria that takes up the secondary dye (safranin) in gram staining procedure

A

Gram- negative bacteria

78
Q

Acid-fast Staining that uses heat in mordanting part. Also referred to as “hot method”

A

Zhiel-Neelsen Method

79
Q

Acid-fast Staining that uses cold in mordanting part. Also referred to as “cold method”

A

Kinyoun Method

-requires only a chemical type of mordant agent which is the phenol

80
Q

Types of culture medium according to composition

A

Synthetic or defined medium

Non-synthetic or complex medium

81
Q

A type of culture medium according to composition in which all the components are KNOWN

A

Synthetic or defined medium

82
Q

Types of culture medium according to composition in which some substances in the medium are UNKNOWN and are also very useful for isolation of medically significant bacteria

A

Non-synthetic or complex medium

e.g. TSB, NB (nutrient broth), and MAC

BACTERIOLOGY (15 decks)

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