Methods of Microbiological Analysis

100問 • 2年前

Butter 張國榮

通報

問題一覧

Which of the following strictly requires pour plate technique?

Enterobacteriaceae/Coliform

Plate Count Agar

Aerobic Plate Count (APC)/ Total Plate Count (TPC), Heterotrophic Plate Count

Yeasts and Molds Count

Potato Dextrose Agar

Violet Red Bile Agar

Enterobacteriaceae/Coliform

Time and Temperature of Bacillus Cereus/Bacillus Subtilis

18-24 h `at 30 C

Time and Temperature of Enterobacteriaceae/Coliform

18-24 h `at 35 C

indicates total viable bacteria in a sample wherein technically all aerobic and mesophilic organisms can grow

Aerobic Plate Count (APC)

sometimes counted separately

yeasts and molds

indicates total viable fungi in sample which implies overall cleanliness in product life cycle; favored in cheese

Yeast and Mold Count

Enterobacteriaceae / Coliform can be further separated into total

total coliform, fecal coliform

indicates hygiene and sanitation

total coliform

conventional test for e.coli

most probable number by multiple tube fermentation technique

ubiquitous aerobic sporeformers commonly assoc with agricultural products

Bacillus spp.

pathogenic; produces cereulide toxin - fried rice syndrome

bacillus cereus

prone to B. cereus

tofu, cereal-based, rice, corn

prone to B. subtilis

bakery products, flour, corn meal

causes spoilage or ropiness in bakery products

B. subtilis

primary beneficial microorganisms in fermented dairy products unique: required minimum level at least 10^6 CFU/mL

Lactic Acid Bacteria

pathogenic aerobic bacteria commonly found in human skin; most common causes of food poisoning

Staphylococcus Aureus

indicates poor hygiene, sanitation, and food handling practices common for many foods esp minimally-processed foods and foods handles after processing

Staphylococcus Aureus

Advantages of Plate-Based Tests

Widely used for enumeration of various foods, Easy-to-perform, with relatively high senstivity and reliability, Selective media allows targeting of specific species in a mixed sample, Allows for isolation and further study of specific microorganisms (by picking colonies)

Plate-based tests have tendencies to underestimate actual counts due to

injured (viable but not culturable) microorganisms, fastidious microorganisms

What makes a microorganism VBNC?

refrigeration, heating, salts/acids

countable range for yeast and mold count

10-150 cfu per plate

countable range for APC

25-250 cfu per plate

Which of the following media is least useful in assessing the quality and safety of Ready-to-Eat Meals?

MRS Agar

High Aerobic Plate Counts will indicate potential quality and safety issues for the following products except:

Yogurt Drink

Erick James consistently pipetted 1.1 mL of inoculum per plate when performing enumerating APC using a conventional spread plate method. What kind of error did they commit?

Blunder

The following are used to quantify fungi in food systems except:

Violet Red Bile Agar

two primary tests/reference microorganism which are conventionally analyzed by MPN

Vibrio spp., Total / Fecal Coliform / E. Coli

pathogenic gram-negative facultative anaerobes assoc with contaminated water and seafood

Vibrio spp.

Quantitative plate-based methods are difficult in Vibrio spp. because

higher salt content and pH, grows quickly, prone to injury causing VBNC

Vibrio spp. uses specialized liquid media to encourage growth while suppressing other microorganisms

alkaline peptone water

Vibrio spp. presumptive positive tubes are inoculated in

Thiosulfate Citrate Bile Salts Agar

V. cholerae : ______ colonies; V parahaemolyticus : blue-green colonies

yellow

V. cholerae : yellow colonies; V parahaemolyticus : ______ colonies

blue-green

gram-negative non sporeforming bacteria which ferment actose at 35-37 C to produce acid and gas

Coliforms

coliforms which can ferment lactose within 48 hours at 45+- 0.5C

Fecal/thermotolerant coliforms

Only considered E.coli if tests result in

dark centered and flat colonies with/without metallic green sheen, gram negative, gas produced in LST, +/+/-/-

media for presumptive test for coliforms, fecal coliforms, and E. coli

Lauryl Sulfate Tryptose (LST) Broth

media for confirmed test for total coliforms

Brilliant Green Lactose Bile (BGLB) Broth

media for completed test for E.coli

Eosin-Methylene Blue (EMB) Agar

Each test require 35C +- 0.5C & 48+- 3h except:

Fecal Coliform

If EMBA-positive what is the next step?

Biochemical test

gold standard method for Vibrio spp. and total coliforms/fecal coliforms/ E.coli with higher sensitivity

MPN Method

- use of hemacytometer, a microscope and a counting chamber, to directly count microscopic cells in suspension yeasts in brewing detection and enumeration of parasites (amoeba, helminths)

Direct Microscopic Counting

turbidimetry is measured through

visual estimation, McFarland standards, turbidimeter, spectrophotometer, OD/ODU

closest in principle to conventional methods

Dry Film Methods

a real-time analysis which reacts with luciferin and luciferase to produce visible light (fluorescence)commonly used for in-line and environmental monitoring

ATP test

more commonly used in the food industry to detect allergens(proteins)

Enzyme-Linked Immunosorbent Assay (ELISA)

generally used in foods to indicate the presence and absence of a specific microorganisms - usually uses a combination of both liquid and solid media

Conventional (Culture-Dependent) Qualitative Methods

In conventional qualitative methods, enrichment steps no longer represent the population by the time of plating

True

Results for repeated inoculation and incubation several times to ensure growth and identification of target microorganism

enrichment steps are no longer representative, microorganisms cannot be enumerated, growth of target = positive

enteric Gram-negative (coliform/Enterobacteriaceae); one of the most significant foodborne pathogens assoc with animal products

Salmonella spp.

What general steps were involved in detecting Salmonella spp.?

Pre-enrichment, Selective enrichment, Isolation, Identification

Pre-enrichment of Salmonella spp. involves

use of non-selective broth of lactose broth, tryipticase soy broth, buffered peptone water, nutrient broth, resuscitates / enriches

to transfer and incubate two selective broth to screen for Salmonella spp.

Selective Enrichment

streak plating from positive selective broths to three selective agar plates

Isolation

The following are three selective agar plates used in Isolation of Salmonella spp. except

Oxford agar (OXA)

incubation of agar plates after isolation of salmonella spp.

24+- 2 h at 35 C

Identification involves the use of various __________ to confirm the identity of Salmonella spp. presumptive.

Biochemical tests

Primary tool for identification of Salmonella

Lysine Iron Agar, Triple Sugar Iron Agar, Urea Broth

Alkali Lysine Iron Agar : purple ; Triple Sugar Iron Agar : ____

red

Acid Lysine Iron Agar : _____ ; Triple Sugar Iron Agar : yellow

purple

Acid Lysine Iron Agar : purple ; Triple Sugar Iron Agar : ____

yellow

Alkali Lysine Iron Agar : _____ ; Triple Sugar Iron Agar : red

purple

Urea broth = negative

yellow

IMViC test result for Salmonella spp.

-/+/-/+

A company producing pasteurized egg white products detected Salmonella spp. through in-line PCR testing but was not abe to confirm its presence through conventional methods. Which of the following is the most likely explanation for this?

Salmonella spp. in the product were already inactivated

an ubiquitous Gram-positive (nonsporeformer) that can exhibit psychrotrophic growth which can grow as low as 4C in refrigerated and temperature-abused frozen foods

Listeria spp.

Pre-enrichment of Listeria spp. includes

non-selective broth of Buffered Listeria Enrichment Broth (BLEB), short incubation (4h) at 30C

selective agents are added aseptically directly into incubated non-selective agar

Selective enrichment of Listeria spp.

selective agents added aseptically into agar in Listeria spp.

10 mg/L acriflavin, 50 mg/L cycloheximide, 40 mg/L sodium nalidixic acid

The following are two non-selective agar plates used in Isolation of Listeria spp. except

PALCAM agar

IMViC test result for Listeria spp.

-/+/+/-

ubiquitous Gram positive anaerobic sporeformer which produces enterotoxins

Clostridium perfringens

sporeformer associated with temperature-abuse meat products, prefers oxygen-free conditions, easily damaged by low temperature conditions

Uneven contamination occur on foods due to anaerobic nature of C. perfringens - ways to prepare sample

Sample entire food, 25 g each from different parts

C. perfringens can rapidly lose viability during refrigeration and freezing

do not freeze, 10 C storage, examine within 8h of collection

Clostridium perfringens Pour plate in ? Incubate anaerobically at 35C for 20-24 hrs Inoculate in Chopped Liver Broth (LB) or Cooked Meat Medium (CMM) Incubate anaerobically at 35C for 24-48 hrs

Tryptose-sulfite-cylcloserine (TSC)

Clostridium perfringens Pour plate in Tryptose-sulfite-cylcloserine (TSC) Incubate anaerobically at 35C for 20-24 hrs Inoculate in ?? or ?? Incubate anaerobically at 35C for 24-48 hrs

Chopped Liver Broth (LB), Cooked Meat Medium (CMM)

In Clostridium perfringens, typical colonies in TSC or LB/CMM and grow in

thioglycollate broth

Clostridium perfringens

black colonies in TSC, non motile, gram + rods, nitrate reduction to nitrite, lactose fermenter, gelatinase positive

Gram-negative thermophilic microaerophile

Campylobacter spp.

Pre-enrichment of Campylobacter spp. includes

Campylobacter enrichment broth (CEB), within 10 days = 37 C for 4 hours, beyond 10 days = 30 C for 2 hours

Campylobacter jejuni can cause

Bloody Diarrhea, Fever 40C

thermohilic and microaerophilic which is highly sensitive to atmospheric oxygen, difficult to culture, and easily goes into VBNC form

Campylobacter spp.

Identification of Campylobacter spp.

wet mounts 1:1 crystal violet:saline

the conventional method for charaterizing and identifying microorganisms

Biochemical Tests

prerequisite step for biochemical tests:

Isolation

most common approach for isolation

streak plating

most common initial test based on cell wall composition

gram reaction

Differential staining uses a series of four reagents

Primary Stain, Mordant, Decolorizer, Secondary Stain

test for catalase enzyme by reacting 3% hydrogen peroxide

catalase test

most aerobic bacteria are

catalase-positive

positive: ________; negative: no change

bubbling

gram positive cocci

catalase test

__________: catalase-positive ; streptococcus spp. : catalase-negative

Staphylococcus spp.

test for cytochrome c by reacting with TMPD; positive = dark purple; gram-negative rods

Oxidase Test

100

pseudomonas spp. : oxidase-positive ; ________ : oxidase-negative

enterobacteriaceae

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問題一覧

Which of the following strictly requires pour plate technique?

Enterobacteriaceae/Coliform

Plate Count Agar

Aerobic Plate Count (APC)/ Total Plate Count (TPC), Heterotrophic Plate Count

Yeasts and Molds Count

Potato Dextrose Agar

Violet Red Bile Agar

Enterobacteriaceae/Coliform

Time and Temperature of Bacillus Cereus/Bacillus Subtilis

18-24 h `at 30 C

Time and Temperature of Enterobacteriaceae/Coliform

18-24 h `at 35 C

indicates total viable bacteria in a sample wherein technically all aerobic and mesophilic organisms can grow

Aerobic Plate Count (APC)

sometimes counted separately

yeasts and molds

indicates total viable fungi in sample which implies overall cleanliness in product life cycle; favored in cheese

Yeast and Mold Count

Enterobacteriaceae / Coliform can be further separated into total

total coliform, fecal coliform

indicates hygiene and sanitation

total coliform

conventional test for e.coli

most probable number by multiple tube fermentation technique

ubiquitous aerobic sporeformers commonly assoc with agricultural products

Bacillus spp.

pathogenic; produces cereulide toxin - fried rice syndrome

bacillus cereus

prone to B. cereus

tofu, cereal-based, rice, corn

prone to B. subtilis

bakery products, flour, corn meal

causes spoilage or ropiness in bakery products

B. subtilis

primary beneficial microorganisms in fermented dairy products unique: required minimum level at least 10^6 CFU/mL

Lactic Acid Bacteria

pathogenic aerobic bacteria commonly found in human skin; most common causes of food poisoning

Staphylococcus Aureus

indicates poor hygiene, sanitation, and food handling practices common for many foods esp minimally-processed foods and foods handles after processing

Staphylococcus Aureus

Advantages of Plate-Based Tests

Plate-based tests have tendencies to underestimate actual counts due to

injured (viable but not culturable) microorganisms, fastidious microorganisms

What makes a microorganism VBNC?

refrigeration, heating, salts/acids

countable range for yeast and mold count

10-150 cfu per plate

countable range for APC

25-250 cfu per plate

Which of the following media is least useful in assessing the quality and safety of Ready-to-Eat Meals?

MRS Agar

High Aerobic Plate Counts will indicate potential quality and safety issues for the following products except:

Yogurt Drink

Erick James consistently pipetted 1.1 mL of inoculum per plate when performing enumerating APC using a conventional spread plate method. What kind of error did they commit?

Blunder

The following are used to quantify fungi in food systems except:

Violet Red Bile Agar

two primary tests/reference microorganism which are conventionally analyzed by MPN

Vibrio spp., Total / Fecal Coliform / E. Coli

pathogenic gram-negative facultative anaerobes assoc with contaminated water and seafood

Vibrio spp.

Quantitative plate-based methods are difficult in Vibrio spp. because

higher salt content and pH, grows quickly, prone to injury causing VBNC

Vibrio spp. uses specialized liquid media to encourage growth while suppressing other microorganisms

alkaline peptone water

Vibrio spp. presumptive positive tubes are inoculated in

Thiosulfate Citrate Bile Salts Agar

V. cholerae : ______ colonies; V parahaemolyticus : blue-green colonies

yellow

V. cholerae : yellow colonies; V parahaemolyticus : ______ colonies

blue-green

gram-negative non sporeforming bacteria which ferment actose at 35-37 C to produce acid and gas

Coliforms

coliforms which can ferment lactose within 48 hours at 45+- 0.5C

Fecal/thermotolerant coliforms

Only considered E.coli if tests result in

dark centered and flat colonies with/without metallic green sheen, gram negative, gas produced in LST, +/+/-/-

media for presumptive test for coliforms, fecal coliforms, and E. coli

Lauryl Sulfate Tryptose (LST) Broth

media for confirmed test for total coliforms

Brilliant Green Lactose Bile (BGLB) Broth

media for completed test for E.coli

Eosin-Methylene Blue (EMB) Agar

Each test require 35C +- 0.5C & 48+- 3h except:

Fecal Coliform

If EMBA-positive what is the next step?

Biochemical test

gold standard method for Vibrio spp. and total coliforms/fecal coliforms/ E.coli with higher sensitivity

MPN Method

- use of hemacytometer, a microscope and a counting chamber, to directly count microscopic cells in suspension yeasts in brewing detection and enumeration of parasites (amoeba, helminths)

Direct Microscopic Counting

turbidimetry is measured through

visual estimation, McFarland standards, turbidimeter, spectrophotometer, OD/ODU

closest in principle to conventional methods

Dry Film Methods

a real-time analysis which reacts with luciferin and luciferase to produce visible light (fluorescence)commonly used for in-line and environmental monitoring

ATP test

more commonly used in the food industry to detect allergens(proteins)

Enzyme-Linked Immunosorbent Assay (ELISA)

generally used in foods to indicate the presence and absence of a specific microorganisms - usually uses a combination of both liquid and solid media

Conventional (Culture-Dependent) Qualitative Methods

In conventional qualitative methods, enrichment steps no longer represent the population by the time of plating

True

Results for repeated inoculation and incubation several times to ensure growth and identification of target microorganism

enrichment steps are no longer representative, microorganisms cannot be enumerated, growth of target = positive

enteric Gram-negative (coliform/Enterobacteriaceae); one of the most significant foodborne pathogens assoc with animal products

Salmonella spp.

What general steps were involved in detecting Salmonella spp.?

Pre-enrichment, Selective enrichment, Isolation, Identification

Pre-enrichment of Salmonella spp. involves

use of non-selective broth of lactose broth, tryipticase soy broth, buffered peptone water, nutrient broth, resuscitates / enriches

to transfer and incubate two selective broth to screen for Salmonella spp.

Selective Enrichment

streak plating from positive selective broths to three selective agar plates

Isolation

The following are three selective agar plates used in Isolation of Salmonella spp. except

Oxford agar (OXA)

incubation of agar plates after isolation of salmonella spp.

24+- 2 h at 35 C

Identification involves the use of various __________ to confirm the identity of Salmonella spp. presumptive.

Biochemical tests

Primary tool for identification of Salmonella

Lysine Iron Agar, Triple Sugar Iron Agar, Urea Broth

Alkali Lysine Iron Agar : purple ; Triple Sugar Iron Agar : ____

red

Acid Lysine Iron Agar : _____ ; Triple Sugar Iron Agar : yellow

purple

Acid Lysine Iron Agar : purple ; Triple Sugar Iron Agar : ____

yellow

Alkali Lysine Iron Agar : _____ ; Triple Sugar Iron Agar : red

purple

Urea broth = negative

yellow

IMViC test result for Salmonella spp.

-/+/-/+

Salmonella spp. in the product were already inactivated

an ubiquitous Gram-positive (nonsporeformer) that can exhibit psychrotrophic growth which can grow as low as 4C in refrigerated and temperature-abused frozen foods

Listeria spp.

Pre-enrichment of Listeria spp. includes

non-selective broth of Buffered Listeria Enrichment Broth (BLEB), short incubation (4h) at 30C

selective agents are added aseptically directly into incubated non-selective agar

Selective enrichment of Listeria spp.

selective agents added aseptically into agar in Listeria spp.

10 mg/L acriflavin, 50 mg/L cycloheximide, 40 mg/L sodium nalidixic acid

The following are two non-selective agar plates used in Isolation of Listeria spp. except

PALCAM agar

IMViC test result for Listeria spp.

-/+/+/-

ubiquitous Gram positive anaerobic sporeformer which produces enterotoxins

Clostridium perfringens

sporeformer associated with temperature-abuse meat products, prefers oxygen-free conditions, easily damaged by low temperature conditions

Uneven contamination occur on foods due to anaerobic nature of C. perfringens - ways to prepare sample

Sample entire food, 25 g each from different parts

C. perfringens can rapidly lose viability during refrigeration and freezing

do not freeze, 10 C storage, examine within 8h of collection

Clostridium perfringens Pour plate in ? Incubate anaerobically at 35C for 20-24 hrs Inoculate in Chopped Liver Broth (LB) or Cooked Meat Medium (CMM) Incubate anaerobically at 35C for 24-48 hrs

Tryptose-sulfite-cylcloserine (TSC)

Clostridium perfringens Pour plate in Tryptose-sulfite-cylcloserine (TSC) Incubate anaerobically at 35C for 20-24 hrs Inoculate in ?? or ?? Incubate anaerobically at 35C for 24-48 hrs

Chopped Liver Broth (LB), Cooked Meat Medium (CMM)

In Clostridium perfringens, typical colonies in TSC or LB/CMM and grow in

thioglycollate broth

Clostridium perfringens

black colonies in TSC, non motile, gram + rods, nitrate reduction to nitrite, lactose fermenter, gelatinase positive

Gram-negative thermophilic microaerophile

Campylobacter spp.

Pre-enrichment of Campylobacter spp. includes

Campylobacter enrichment broth (CEB), within 10 days = 37 C for 4 hours, beyond 10 days = 30 C for 2 hours

Campylobacter jejuni can cause

Bloody Diarrhea, Fever 40C

thermohilic and microaerophilic which is highly sensitive to atmospheric oxygen, difficult to culture, and easily goes into VBNC form

Campylobacter spp.

Identification of Campylobacter spp.

wet mounts 1:1 crystal violet:saline

the conventional method for charaterizing and identifying microorganisms

Biochemical Tests

prerequisite step for biochemical tests:

Isolation

most common approach for isolation

streak plating

most common initial test based on cell wall composition

gram reaction

Differential staining uses a series of four reagents

Primary Stain, Mordant, Decolorizer, Secondary Stain

test for catalase enzyme by reacting 3% hydrogen peroxide

catalase test

most aerobic bacteria are

catalase-positive

positive: ________; negative: no change

bubbling

gram positive cocci

catalase test

__________: catalase-positive ; streptococcus spp. : catalase-negative

Staphylococcus spp.

test for cytochrome c by reacting with TMPD; positive = dark purple; gram-negative rods

Oxidase Test

100

pseudomonas spp. : oxidase-positive ; ________ : oxidase-negative

enterobacteriaceae