(P) Lec 4: Culture Staining (Part 1) Flashcards by Kchianna Lauren Coronel

Flow of Lab Procedures for the Diagnosis of Infectious Diseases

This is the first step upon receiving the specimen

Direct Examination by Microscopy

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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?

Feces

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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

Gram (-) bacilli

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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

Gram (+) bacteria

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Flow of Lab Procedures for the Diagnosis of Infectious Diseases

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

Dysentery (infection in your intestines that causes bloody diarrhea)

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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

Culture

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Flow of Lab Procedures for the Diagnosis of Infectious Diseases

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

Plated agar

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Flow of Lab Procedures for the Diagnosis of Infectious Diseases

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

Urethral discharge

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Flow of Lab Procedures for the Diagnosis of Infectious Diseases

Urethral discharge is put into a tube containing what broth?

Thioglycollate

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Flow of Lab Procedures for the Diagnosis of Infectious Diseases

How many hours must cultures be isolated for?

16 to 24

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Flow of Lab Procedures for the Diagnosis of Infectious Diseases

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

Last part where you swabbed

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

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Flow of Lab Procedures for the Diagnosis of Infectious Diseases

This step is done after culturing your specimen

Analysis of Cultivated Organisms (identification and susceptibility testing)

e.g. biochemical testing for specific pathogens

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Flow of Lab Procedures for the Diagnosis of Infectious Diseases

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

Simultaneously (at the same time)

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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

Microscopy

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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

Preliminary
Visualization
Different
Cultivated

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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

Inflammation
Preculture & Grow
Characterization

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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?

Mycobacterium tuberculosis

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Applications of Microscopy

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

Weeks

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Applications of Microscopy

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

PMNs = 25
Epithelial Cells = 10

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

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Applications of Microscopy

What is the gold standard in identifying bacterial species?

Culture and sensitivity

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Applications of Microscopy

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

Workups that are to be done (set of tests)

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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

Bright Field Microscope (normal microscope)

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Microscopes

How do you compute for total lens magnification?

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

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Microscopes

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

Resolving Power

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# 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

1. Blue & Wavelength 2. Condenser 3. Diaphragm 4. Immersion

# Microscopes This is used in staining techniques

Contrast

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

80%

# 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

Phase Contrast Microscope

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

False (staining is not needed)

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

Phase Contrast

# 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

Fluorescent Microscopy

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

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

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

Dye and light filter used

# Microscopes What are the 3 common dyes used in fluorescent microscopy?

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

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

Blue

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

Violet | Commonly used on fungi

# 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.

Dark Field Microscopy

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

Spirochetes (are very thin)

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

A dark field

# Microscopes TOF: Staining is not required in dark field microscopy

True

# 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

Electron Microscopy

# Microscopes Electron microscopy is usually used for what organisms?

Viruses (as they are very small)

# Microscopes What is the magnification of the electron microscope?

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

# Types of Electron Microscopes This passes the electron beam through objects and allows visualization of its internal structure

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

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

Scanning Electron Microscope (SEM) | Clue: (S)urface structures = (S)canning electron microscope

# 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?

Hanging drop method

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

Hanging drop method

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

False (put the sample on the cover slip)

# Staining Techniques This technique uses a single stain to color a bacterial cell

Simple Staining

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

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

# Staining Techniques Staining can determine what characteristic of the bacteria? Clue: cocci, bacilli, etc.

Morphology

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

The background

# 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

Corynebacterium

# Staining Techniques What does the pleomorphism of Corynebacterium mean?

Irregular shapes

# Staining Techniques What color are the metachromatic granules of Corynebacterium?

Reddish-purple

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

Volutin/Metachromatic Granules

# Staining Techniques What is the arrangement of Corynebacterium?

Palisading/Picket-fence

# Staining Techniques What are the 3 simple stains shown in the powerpoint?

1. Methylene blue 2. Basic fuchsin 3. Crystal violet

# 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.

Negative Staining

# Staining Techniques What kind of dyes are used in negative staining?

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

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

Capsular Staining

# Staining Techniques Match the following for capsular staining: 1. Reagent 2. Decolorizer 3. Counterstain A. Crystal violet B. 20% copper sulfate

1. A 2. B 3. B

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

Light blue (OR a white halo)

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

Hans Christian Gram

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

Gram Staining

# Staining Techniques Culture and Sensitivity will produce results in how many days?

3-5 days

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

Cell wall and composition

# Staining Techniques When staining, you usually perform this step first

Heat fix or air-dry

# Staining Techniques TOF: All stains can be heat fixed

False (e.g. endospore stains and capsular stains cannot as it could destroy the capsule) | These only need to be air dried

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

An insoluble complex

# Staining Techniques TOF: Acetone is also a decolorizer

True

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

Gram (+)

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

Gram (-)

# 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?

Gram Variable Bacteria

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

False (waxy, not slimy) | e.g. Acid-Fast Organisms like Nocardia and Mycobacterium

# 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

1. 18 hours 2. Thin 3. Decolorization

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

False (gram positive to negative or variable)

# 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

False (over application)

# Staining Techniques Used for bacillus and clostridium | Clue: Think about what classification these 2 belong in

Spore Staining

# Staining Techniques These 2 bacteria form endospores

Bacillus and Clostridium

# 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)

Endospores

# Staining Techniques When do Bacillus and Clostridium form endospores (dormant form)?

When their energy is depleted

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

Their vegetative states

# 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

Schaeffer-Fulton Method | Stains the endospore green

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

Dorner Method

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

Acid-Fast Staining

# Staining Techniques The mycolic acid of AFB are resistant to what?

Decolorization

# Staining Techniques What are the 3 AFBs?

1. Mycobacterium spp. 2. Nocardia spp. 3. Cryptosporidium spp.

# 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

Cold Method/Kinyoun Acid Fast

# 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

Hot Method/Ziehl Neelsen Acid Fast Method

# Staining Techniques Carbolfuchsin contains what?

Phenol (an alcohol)

# Staining Techniques This is a corrosive combustible poison and should be handled carefully

Phenol (an alcohol)