(P) Lec 4: Culture Staining (Part 2) Flashcards by Kchianna Lauren Coronel

Cultivation Methods

Fill in the blanks:
Consider the following factors:
1. Choice of suitable (blank)
2. Isolation of bacterial organism in (blank) culture

Medium
Pure

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Cultivation Methods

The process of growing microorganisms in culture by taking bacteria from an infection site

Bacterial Culture (the gold standard)

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Cultivation Methods (main purposes)

Fill in the blanks:
To grow and isolate all bacteria present in a (blank) specimen

Clinical

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Cultivation Methods (main purposes)

Fill in the blanks:
To determine which of the bacteria are most likely causing a/an (blank) and which are contaminants/colonizers

Infection

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Cultivation Methods (main purposes)

Fill in the blanks:
To obtain sufficient growth of clinically (blank) bacteria to allow identification, characterization, and susceptibility testing

Relevant

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Cultivation Methods

This depends on the availability of essential nutrients & appropriate environmental conditions

Bacterial Survival

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Cultivation Methods

TOF: Bacteria will grow once plated

False (recall the optimum conditions for growth)

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Phase of Growth Media

Are nutrients dissolved in water
Turbidity/cloudiness is a positive result for the presence of bacteria

Liquid or Broth

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Phase of Growth Media

How much of the bacterial concentration is needed for turbidity to be detected in the liquid/broth media?

10^6 bacteria/mL

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Phase of Growth Media

A combination of a solidifying agent added to the nutrients and water

Solid or Agar

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Phase of Growth Media

What is the most common solid/agar media?

Agarose

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Phase of Growth Media

What is the melting temperature and re-solidification temperature of agarose?

Melting: >95ºC
Re-solidification: <50ºC

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Phase of Growth Media

This phase of the medium comprises of a semi-solid characterization, what is it called?

Biphasic/Intermediate Phase (for bacterial motility)

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Media Classification and Functions

This comes with specific nutrients that will enhance pathogen growth

Enrichment Media

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Media Classification and Functions

Identify the media:
Examples are BCYA (with lysine), Thioglycollate, LIM Broth, and GN Broth

Enrichment Media

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Media Classification and Functions (Enrichment Media)

Buffer Charcoal Yeast Agar (BCYA) with lysine in the Enrichment Media supports the growth of what bacteria?

L. pneumophilia (lysine supports the growth of this)

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Media Classification and Functions (Enrichment Media)

Thioglycollate supports the growth of what bacteria?

Anaerobes (because thioglycollate and cystine remove O2)

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Media Classification and Functions

This contains agents that are inhibitory to all organisms except those selected for specific growth

Selective Media

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Media Classification and Functions (Selective Media)

PEA supports/allows the growth of what bacteria?

Gram (+) bacteria

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Media Classification and Functions (Selective Media)

Thayer-Martin supports/allows the growth of what bacteria?

Neisseria gonorrhea

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Media Classification and Functions

This supports the growth of non-fastidious organisms (only the basic needs for growth)

Nutritive/Supportive Media

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Media Classification and Functions (Nutritive/Supportive Media)

Tripticase Soy Agar (TSA), Nutrient Agar, and Sabouraud Dextrose Agar (SDA) supports the growth of what bacteria?

Most organisms

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Media Classification and Functions (Nutritive/Supportive Media)

This bacteria requires basic needs only

Non-Fastidious Organisms

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Media Classification and Functions (Nutritive/Supportive Media)

This bacteria requires specific/complex requirements for growth

Fastidious Organisms

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# Media Classification and Functions This allows bacteria to exhibit certain metabolic or cultural characteristics that can be used to distinguish them from other bacteria (e.g. MAC and BAP)

Differential Media

# Media Classification and Functions (Differential Media) This is both a differential (demonstrates whether the organisms are lactose fermenters or not) and selective (for gram negative) media

MacConkey Agar (MAC)

# Media Classification and Functions (Differential Media) What is the positive indicator for the presence of lactose fermenters in MAC agar?

Pink colonies

# Media Classification and Functions (Differential Media) What is the negative indicator for the presence of lactose fermenters in MAC agar?

Colorless colonies

# Media Classification and Functions (Differential Media) This is a nutritive and differential media that supports the growth of most fastidious organisms and a differential media that demonstrates the hemolytic pattern of bacteria

Blood Agar Plate (BAP)

# Media Classification and Functions (Differential Media) Some microorganisms have this enzyme which hemolyzes blood and can be seen in the agar plate

Hemolysin

# Plating Media This nutritive media supports the growth for all and most fastidious bacteria and demonstrates a hemolytic pattern

Sheep Blood Agar | Has 5% defibrilgenated sheep's blood

# Plating Media (Sheep's Blood Agar) Refers to the complete clearing of RBCs around the bacterial colony

Beta hemolysis

# Plating Media (Sheep's Blood Agar) Refers to greenish discoloration

Alpha hemolysis

# Plating Media (Sheep's Blood Agar) Refers to no hemolysis

Gamma hemolysis

# Plating Media Uses hemolyzed blood and supports the growth of fastidious bacteria (same as BAP)

Chocolate Agar Plate

# Plating Media (Chocolate Agar Plate) The hemolyzed blood releases 2 factors which support the growth of fastidious bacteria

Factor X (hemin) and Factor V (NAD) | e.g. Haemophilus spp. and Neisseria gonorrhea

# Plating Media This has colistin and nalidixic acid (antibiotics) which inhibits gram-negative bacteria and selective for gram-positive bacteria

Columbia (SNA) with Blood Agar

# Plating Media This is both a selective agar and differential agar with crystal violet as an inhibitor for gram (+) bacteria and fungi

MacConkey Agar

# Plating Media (MacConkey) What is the indicator for MAC after crystal violet has inhibited the growth of gram-positive bacteria and fungi?

Neutral Red (pH indicator) | When there is a change in pH, there'll be a change in the indicator too

# Plating Media (MacConkey) When there is bacterial fermentation of lactose, there is a production of acid which manifests as what result?

Pink Colonies | Due to decreased pH

# Plating Media This has bile salts and dyes as the inhibitors (bromothymol blue and acid fuchsin)

Hektoen Enteric Agar (HEA)

Hektoen Enteric Agar (HEA) is the selective media for what 2 bacteria?

Salmonella and Shigella | + other gram positive bacteria

# Plating Media (Hektoen Enteric Agar) For the differential factor, non-enteric pathogens will not grow and will appear as what color?

Orange to salmon-colored

# Plating Media (Hektoen Enteric Agar) This can differentiate between what 2 kinds of organisms involving lactose?

Lactose-producing and non lactose-producing

# Plating Media (Hektoen Enteric Agar) TOF: This uses gram positive organisms

False (gram negative)

# Plating Media (Hektoen Enteric Agar) This can differentiate between what 2 kinds of organisms involving hydrogen sulfide?

H2S producer and non-H2S producer

# Plating Media (Hektoen Enteric Agar) What is the pH indicator?

Bromthymol blue

# Plating Media (Hektoen Enteric Agar) Presence of lactose indicates many changes in colonies, what is manifested when there are no lactose-fermenters?

No color change

# Plating Media (Hektoen Enteric Agar) What is the indicator for H2S (hydrogen sulfide) and what will be the color of the colonies?

Ammonium Citrate; Black

# Plating Media This is selective and differential for Shigella spp. and Salmonella spp.

Xylose Lysine Desoxycholate (XLD) Agar

# Plating Media (Xylose Lysine Desoxycholate) What is the inhibitor for XLD?

Salt and sodium desoxycholate

# Plating Media (Xylose Lysine Desoxycholate) What is the pH indicator for XLD?

Phenol

# Environmental Requirements Aerobic bacteria have the following enzymes: catalase, superoxide dismutase, and peroxidase. What is its cumulative function?

Conversion of the toxic form of oxygen (breakdown of hydrogen peroxide into water and oxygen)

# Environmental Requirements Most clinically relevant bacteria belong to 3 categories which are?

1. Aerobic 2. Facultative anaerobes 3. Obligate anaerobes

# Environmental Requirements These organisms: Neisseriaceae, Pseudomonas spp., Brucella, Bordetella, and Francicella all belong to what bacterial category of oxygen and CO2 availability?

Aerobes

# Environmental Requirements These bacteria can live with or without oxygen

Facultative anaerobes

# Environmental Requirements These bacteria require very little or no oxygen at all

Obligate anaerobes | e.g. Clostridium tetani

# Environmental Requirements These are organisms that thrive in the presence of high concentrations of carbon dioxide

Capnophilic organisms

# Environmental Requirements What is the catalyst for the GASPAK anaerobic jar?

Palladium pellets

# Environmental Requirements What is the aerobic indicator for the GASPAK anaerobic jar?

Methylene blue

# Environmental Requirements What is the anaerobic indicator for the GASPAK anaerobic jar?

Colorless

# Environmental Requirements How many hours does it take to definitively conclude that an anaerobic environment exists in the GASPAK jar (methylene blue indicator)?

2 hours | If it's still blue after 2 hours, then it is not anaerobic

# Environmental Requirements This method will consume most of the O2 present inside producing elevated levels of CO2 (used for microaerophilic/capnophilic organisms)

Candle Jar

# Environmental Requirements In the thioglycollate tube, the turbidity of a tube with obligate aerobes will cluster in what portion of the tube?

Top of the tube

# Environmental Requirements In the thioglycollate tube, the turbidity of a tube with obligate anaerobes will cluster in what portion of the tube?

Bottom of the tube

# Environmental Requirements In the thioglycollate tube, the turbidity of a tube with facultataive anaerobes will cluster in what portion of the tube?

Throughout the media but more on the top portion

# Environmental Requirements In the thioglycollate tube, the turbidity of a tube with aerotolerant anaerobes will cluster in what portion of the tube?

Throughout the medium

# Environmental Requirements What is the ideal temperature?

Body temperature or 37ºC (incubators maintain it at 35-37ºC) | Some require higher (42ºC) while some require lower (20-40ºC)

# Environmental Requirements What organism requires a higher temperature (42ºC acc. to the video)?

Campylobacter

# Environmental Requirements What organism requires a lower temperature (20-40ºC acc. to the video)?

Listeria monocytogenes

# Environmental Requirements Most clinically relevant bacteria prefer a near neutral pH range, which is?

6.5 to 7.5

# Environmental Requirements TOF: Abundance of water affects the metabolic pathways in a negative manner

False (loss of water; dehyrated media)

# Environmental Requirements An increase in solute concentration in the media can lead to what? (2 answers)

Osmotic Shock and Cell Lysis

# Environmental Requirements Sealing the agar plates is followed by what step?

Using humidified incubators

# Plating Technique This uses a diluted suspension of cells mixed with melted agar at 50ºC and poured in a petri dish; the diluted suspension contains separated colonies where you can pick a colony of desired type

Pour Plate Technique

# Plating Technique The suspension is streaked on the surface of the agar plate with a wire loop that is heated in between streaks (most commonly perfomed in the lab)

Streak Plate Technique

# Plating Technique In Streak Plate Technique, as streaking continues, what happens to the cells?

Fewer and fewer cells remain on the wire loop (pinpoint/discrete colonies)

# Plating Technique In Streak Plate Technique, this streak may deposit single cells on the agar, which streak is it?

Final streak

# Plating Technique The volume of a dilute microbial suspension is transferred to the center of the agar plate and is spread evenly using sterile bent rod | Can be used in urine culture but that mostly uses a calibrated wire loop

Spread Plate Technique

# Plating Technique In Spread Plate Technique, the # of viable colonies equals what?

No. of viable organisms

# Plating Technique TOF: The Spread Plate Technique cannot be used to count the microbial population

False (you can)

# Isolation of Microorganisms in Pure Culture There is serial dilution of the sample that will be plated and cultured afterwards but this is less ideal

Dilution