FMLec | M2 Culture-Dependent Techniques, APC & EAPC Calculations

2 general ways to detect organisms in food

• Culture-dependent techniques

• Non-culture-dependent techniques

11 culture-dependent techniques

ct3m adrd mm

1. Conventional standard plate counts (SPC/APC)

2. Turbidimetric measurement

3. Most probable number (MPN)

4. Membrane filtration

5. Microscope colony count

6. Agar droplet count

7. Dye reduction

8. Roll tubes

9. Dry film and related methods

10. Microbiological examination of surfaces

11. Metabolically injured microorganisms

Culture-dependent technique

• _ is also known as aerobic plate count (APC)

• Used to count the number of viable cells (i.e., those capable of forming an offspring) and thus the number of colony forming units (CFU) because the assumption is each colony arises from 1 viable cell

Conventional standard plate count (SPC)

Why do we count CFUs in SPCs?

It’s because of the assumption that each viable cell can grow and divide to yield one colony, 1 cell = 1 colony

When is APC or SPC used?

For counting the no. of viable cells = no. of CFUs since 1 cell = 1 colony

7 factors affecting viable count (in A/SPC)

sdn ppti

• Sampling methods employed

• Distribution of organisms in the food sample

• Nature of food biota and food material

• Pre-examination history of the food product

  • Allows us to know the (1) relative number of organisms in food sample, (2) existence of other competing or antagonistic organisms

• Plating medium used

  • Nutritional adequacy, pH, oxidation-reduction potential (Eh) of the plating medium, water activity npow

• Type of diluent

• Incubation time and temperature used

Explain SPC procedure

1. Food sample homogenization

2. Serial dilution

3. Plating in or onto a suitable agar medium

4. Incubation at appropriate temperature (for specific period of time)

5. Counting of colonies

6. Calculation of CFU/g or CFU/mL

2 equipments which may be used for food sample homogenization

• Stomacher

• Mechanical blender

• _ homogenizes specimen in a special plastic bag

• Vigorous pounding of 2 paddles that shear the food specimen

• Microorganisms are then released into the diluent

Stomacher

4 advantages of stomacher over mechanical blender

mens

• More pleasant noise level

• Easy to clean

• No heat buildup

• Allows for the storage of homogenates in the freezer

_ is a stepwise process of reducing the concentration of microorganisms in a sample

Serial dilution

Explain the principle behind serial dilution and the precautionary measure prior to doing it

• Serial dilution will thin out microbial population until there is only 1 cell left in a tube of diluent (10-fold decrease in no. of cells as dilution increases)

• Prior to transferring inoculum from 1 tube to another, it’s important to vigorously shake the tube where you’ll be taking your inoculum from, specifically shaking it 25 times in 1 ft arc within 7s or use a vortex mixer

3 major sources of breast milk microbiome

• Child’s oral cavity

  • Presence of oral bacteria in breast milk (e.g., Streptococcus salivarius, S. mitis, Gemella, Rothia)

  • Reverse flow of milk from infant’s mouth → woman’s milk ducts

• Mother’s skin

  • Presence of commensal bacteria (e.g., S. epidermidis, Corynebacterium, Malassezia)

  • Colonization of mammary gland with mother’s skin microbiota thru nipple

• Mother’s digestive tract

  • Presence of anaerobic bacteria of GI tract (e.g., Bifidobacteria, Bacteroides, Clostridium, Saccharomyces)

  • Entero-mammary route during pregnancy and lactation

In plating step (in or onto a suitable agar medium), 2 factors must be considered including _

• Type of media (based on composition and use)

• Plating technique

Types of media based on composition

• Defined media

  • Exact quantitative and qualitative composition

  • Prepare by adding precise amounts of highly purified organic or inorganic chemicals to distilled water

  • Simple = if it contains only 1 carbon (C) source

  • Complex = if more than 1 C source

• Complex media

  • Contain digests of microbial, animal, or plant products, or any number of other highly nutritious yet impure substances

  • e.g.,

    • Casein (milk)

    • Beef extract

    • Peptone (protein hydrolysate)

    • Tryptic soy broth (soybeans)

    • Yeast extract

Explain the types of media based on use/purpose

• General purpose media (e.g., Nutrient Agar)

  • Supports almost all microbial growth

  • Do not contain inhibitory substances

• Enriched media (e.g., Blood Agar)

  • Growth stimulants, e.g., blood, serum, other highly nutritious substances bso

  • Increases the number of desired microorganisms to a detectable level but does not suppress the growth of other bacteria

  • For growing fastidious microorganisms (i.e., those with complex nutritional requirements)

• Selective media (e.g., Mannitol Salt Agar)

  • Inhibits growth of unwanted microorganisms and encourages growth of a particular organism

  • Contains inhibitors, e.g., antibiotics, dyes, toxic compounds, detergents adtd

• Differential media (e.g., Eosin Methylene Blue Agar)

  • Distinguishes different species of bacteria, usually containing indicators (e.g., dyes, pH indicators) that reveal differences in microbial metabolism and enzymatic activity

Type of media based on purpose/use

• Contains growth stimulants, e.g., blood, serum, other highly nutritious substances

• For growing fastidious microorganisms

• Increases the number of desired microorganism to a detectable level but does not suppress the growth of other bacteria

Enriched media

Type of media based on purpose/use

• Distinguishes different species of bacteria

• Usually contains indicators (e.g., dyes) that reveal differences in microbial metabolism and enzymatic activity

Differential media

Type of media based on purpose/use

• Supports almost all microbial growth

• Does not contain any inhibitory substances

General purposed media

Type of media based on purpose/use

• Contains inhibitory substances (e.g., antibiotics, dyes, toxic compounds, detergents)

• Inhibits the growth of unwanted microorganisms while encouraging the growth of a particular organism

Selective media

T/F: Enriched media contains inhibitory substances

FALSE

While enriched media contains growth stimulants, it only serves to increase the number of desired microorganism to a detectable level but does not suppress growth of other bacteria

Type of media based on purpose/use

• e.g., Eosin Methylene Blue Agar (EMBA)

Differential media

Type of media based on purpose/use

• e.g., Nutrient Agar

General purpose media

Type of media based on purpose/use

• e.g., Mannitol Salt Agar

Selective media

Type of media based on purpose/use

• e.g., Blood Agar

Enriched media

Defined or Complex media?

Complex media (with yeast extract, peptone)

Defined or Complex media?

Simple defined media (only 1 C source)

Type of media (based on composition) with exact quantitative and qualitative composition, prepared by adding precise amounts of highly purified organic or inorganic chemicals to dH2O

Defined media

Defined media with only 1 C source

Simple defined media

Defined media with more than 1 C source

Complex defined media

3 plating techniques

• Spread plating (0.1 mL or 100 uL inoculum)

• Pour plating (1 mL or 1000 uL)

• Miles and Misra / Drop Count technique (0.01 mL or 10 uL)

Explain steps to spread plating

1. Pipette 0.1 mL (100 uL) sample into solidified agar surface of plate

2. Spread sample evenly over surface using L rod and turn table

3. Incubate

4. Colonies grow on surface

Explain steps to pour plating

1. Pipette 1 mL (1000 uL) sample into plate

2. Pour molten / liquefied agar

3. Swirl to mix (do figure-8 pattern)

4. Incubate

5. Colonies grow on surface and subsurface

Explain steps to Miles and Misra / Drop Count technique

1. Serially dilute sample

2. Divide plate into 6 parts (or into how many dilutions you’ll plate)

3. Pipette 0.01 mL (10 uL) of sample of particular dilution into each division of plate

4. Incubate

5. Colonies grow on surface

Spread vs. Pour vs. Miles and Misra / Drop Count Technique

cbd tvma	Spread plate	Pour plate	Miles and Misra
Culture media	Pre-solidified media	Molten/liquefied media	Pre-solidified agar
Basis of isolation	Serial dilution, Spatial separation	Serial dilution, Spatial separation	Serial dilution
Desired microorganisms	Present at higher level than any other microorganism	Present at higher level than any other microorganism	Present at higher level than any other microorganism
Type of colonies	Surface	Surface/subsurface	Surface
Vol of inoculum	0.1 mL (100 uL)	1 mL (1000 uL)	0.01 mL (10 uL)
Main lab tool	L rod/Bent glass rod/hockey sticks, Micropipette, Turntable	Micropipette, Water bath (for keeping molten agar liquefied)	Micropipette
Application	Isolation, Enumeration (Heat-sensitive aerobes)	Isolation, Enumeration (Heat-tolerant aerobes, Microaerophiles)	Enumeration (Heat-sensitive aerobes)

_ refer to the 3 key temperature points that define the growth range of a microorganism

Cardinal temperatures

_ refers to the highest temperature at which microbial growth can occur; beyond this, protein denaturation, collapse of cytoplasmic membrane, and thermal lysis may occur pct

Maximum temperature

_ refers to the temperature at which growth rate is fastest and enzymatic reactions occur at maximal possible rate

Optimum temperature

_ refers to the lowest temperature at which microbial growth can occur; beyond this, membrane gelling and transport processes become so slow that growth cannot occur

Minimum temperature

Different types of organisms based on their preferred temperature range

• Psychrophiles = 4 C Polaromonas vacuolata

  • Psychrotolerant

• Mesophiles = 39 C E. coli

• Thermophiles = 60 C Geobacillus stearothermophilus

  • Thermotolerant

• Hyperthermophiles = 88 C Thermococcus celer, 106 C Pyrolobus fumarii

A microbe incubated beyond its maximum (cardinal) temperature may experience _

pct

• Protein denaturation

• Collapse of the cytoplasmic membrane

• Thermal lysis

A microbe incubated at lower its minimum (cardinal) temperature may experience _

membrane gelling and transport processes may be so slow that growth cannot occur

In a microbe incubated at its optimum temperature, _ may be observed

enzymatic reactions occurring at maximal rate

T/F: Mesophiles can grow at 0°C but will have significantly slower growth rates

FALSE

Mesophiles generally cannot grow at 0°C. Psychrotrophs and psychrophiles, however, can.

T/F: Psychrophiles and psychrotrophs can both grow at low temperatures, but only psychrotrophs can tolerate higher temperatures beyond 20°C

TRUE
Psychrotrophs have a broader temperature range and can tolerate higher temperatures, unlike psychrophiles, which die at warmer temperatures.

Explain 5 different types of microorganisms based on oxygen requirements

• Obligate aerobes (Micrococcus luteus)

  • Need O2 to survive

• Facultative anaerobes (E. coli)

  • Can survive without O2 but prefers it when available

• Aerotolerant (Streptococcus mutans)

  • Do not use O2 but can tolerate it

• Strict anaerobes (Methanobacterium formicicum)

  • Exposure to O2 is toxic

• Microaerophiles (Spirillum volutans)

  • Require O2 at low concentrations but atmospheric O2 is harmful to them

T/F: A facultative aerobe and a facultative anaerobe refer to two different types of organisms

FALSE

These terms are used interchangeably; they both refer to organisms that can survive without O2 but prefers it

T/F: Aerotolerant anaerobes and facultative anaerobes both grow in oxygen, but only facultative anaerobes use it for metabolism

TRUE

Facultative anaerobes use aerobic respiration when oxygen is available, while aerotolerant anaerobes never use oxygen but tolerate it.

T/F: All microaerophiles can survive at normal atmospheric oxygen levels (21%)

FALSE

They require low oxygen levels (~2-10%) and are harmed by high oxygen concentrations.

Where to incubate

• Aerobes, facultative anaerobes, aerotolerant

Normal incubator

Where to incubate

• Microaerophiles

Microaerophilic jar or can (has candle inside to keep O2 levels low)

Where to incubate

• Strict anaerobes

• Anoxic jar

• Anoxic glove box

• Make-shift jar with oxygen absorber inside

Methods that may be used for colony counting

• Colony counter equipment

• Mobile applications

• Manually using pen

3 important guidelines in colony counting

1. Count all colony forming units (CFUs), including pinpoint

2. Count immediately after incubation period

3. Store plates at 4C for not more than 24 hrs if it’s impossible to count at once

Describe what can is considered a normal plate

• Spreader-free plate

• Most statistically valid counts

  • Bacteria = 25 - 250 (30 - 300)

  • Fungi = 15 - 150

Why do we consider normal plates?

• This is because counts outside the normal range may be erroneous in the sense that,

  • Dilution factors may exaggerate the low counts

  • Crowded plates may be too difficult to count or may inhibit the growth of some bacteria

6 important rules in calculating and reporting CFU/g or CFU/mL

• Compute values from duplicate plates

• CFU/mL = liquid samples

• CFU/g = solid samples

• Report only the first 2 sig figs

• Express in scientific notation

• Round off to 2 sig figs only at the time of conversion to APC

Explain the rules of rounding off in calculating CFU/mL or CFU/g

1. Round up when 3rd digit > 5

   1. 13,800 = 14 000 = 1.4 ×10^4

2. Round down when 3rd digit < 5

   1. 11,300 = 11,000 = 1.1×10^4

3. When 3rd digit = 5,

   1. UP if 2nd digit is odd

      1. 17,500 = 18,000 = 1.8×10^4

   2. DOWN if 2nd digit is even

      1. 18,500 = 18,000 = 1.8×10^4

Explain the formula for computing original cell density if only 1 dilution gives a valid count

• Cell density = CFU/mL or CFU/g

• CFU/mL or CFU/g = average no. of colonies / original sample volume

• Original sample volume = (D) (vol plated)

Additional rule for computing original cell density

• How do you compute for original cell density if in a duplicate plate set, one falls within the normal range and the other outside?

• Solve figure shown

• Use only the normal counts

• CFU/g = avg no. of colonies / original sample vol

• CFU/g = avg no. of colonies / (D) (vol plated)

Additional rule for computing original cell density

• How do you compute for original cell density if 2 consecutive dilutions give a valid count?

• Solve figure shown

• CFU = total C in plates with valid counts / [(n1 × 1) + (n2 × 0.1)] (D1) (vol plated)

Original cell density

• Solve figure shown

Comprehension check

• Solve the ff.

_ is reported if there are no normal plates

Estimated Aerobic Plate Count (EAPC)

5 instances when EAPC is used

2n spl

1. Number of CFU per plate for all dilutions exceeds 250

2. Number of CFU per plate for all dilutions is less than 25

3. Spreaders

4. Plates with no CFU

5. Laboratory accident

Explain EAPC guidelines when number of CFU per plate for all dilutions exceed 250

• Record the counts as TNTC

• Count CFU from plate closest to 250

• Count CFU in portions of the plate representative of colony distribution

• If there are fewer than 100 colonies / cm²

  • CFU / mL = total no. of colonies in plate / original sample vol

  • CFU / mL = total no. of colonies in plate / (D) (vol plated)

• If there are more than 100 colonies / cm²

  • CFU / mL = (>100) (DF) (area of the plate)

EAPC Calculation

• How do you compute for EAPC CFU if the no. of CFU per plate for all dilutions exceed 250 but less than 100 / cm²?

• Solve figure shown

EAPC CFU / mL = total no. of colonies in plate / (D) (vol plated)

EAPC Calculation

• How do you compute for EAPC CFU if the no. of CFU per plate for all dilutions exceed 250 but more than 100 / cm²?

• Solve figure shown

EAPC CFU / mL = >100 (DF) (area of the plate)
area of plate = 65 cm²

When do we use EAPC computation in the case of spreaders?

• When the area covered by spreaders (including total area of repressed growth) exceeds 50% of plate area

• When the area of repressed growth exceeds 25% of plate area

Enumerate the different types of spreaders

• Chain of colonies formed from disintegration of bacterial clump

• Colonies formed in film of water between agar and bottom of plate

• Colonies formed in film of water on the edge or at the surface of agar

It must be marked _ to denote estimation from counts outside 25 to 250 per plate range

EAPC

EAPC computation

• How do you compute for the EAPC of plates with spreader?

• Count each of 3 distinct spreader types as 1 source

• If only 1 chain exists, count it as 1 colony

• If more than 1 chain appears to originate from separate sources, count each source as 1 colony

• Combine spreader count + colony count to compute for EAPC

EAPC computation

• How do you compute when all plates yield < 25 CFU or no. of CFU per plate for all dilutions is less than 25?

• Solve figure shown

EAPC CFU/mL = < 25 ( 1 / original sample volume)

EAPC CFU/mL = < 25 ( 1 / (D) (vol plated) )

EAPC computation

• How do you compute for EAPC when all plates yield no CFU or all plates from all dilutions have no colonies?

EAPC CFU/mL = < 1 (lowest dilution used)

When plates are known to be contaminated or unsatisfactory, mark it as _ to denote laboratory accident

EAPC

Properties of a good diluent for homogenizing food samples

onn

• Osmotic balance = should match the osmotic pressure of microbial cells to prevent cell lysis or dehydration

• Non-toxic to microorganisms = should not inhibit microbial growth to allow isolation, enumeration

• No or minimal nutritional value = should not support microbial growth before plating, to prevent overgrowth