FMLec | M2 Culture-Dependent Techniques pt. 2

Culture-dependent technique

• Involves rotating plate containing a suitable agar medium

• Uses mechanical device that distributes liquid inoculum on the surface

• Has dispensing arm that moves from near center of plate to the edge (Archimedes spiral)

Spiral plating

T/F: Spiral plating makes use of a mechanical device with a dispensing arm that moves in an Archimedes spiral manner, that is, from the edge to the near center of the plate

FALSE

Spiral plating makes use of a mechanical device with a dispensing arm that moves in an Archimedes spiral manner, that is, from near the center of the plate toward the edge

What is the expected colony development or distribution in spiral plating?

High density cells to be deposited near the center of the plate, then cells become progressively fewer toward the edge

• Rapid and widely used approach to estimating cell numbers in a solution

• Relies on the principle that light is scattered in proportion to the number of (unicellular) cells

• As no. of cells increases, the amount of light scattered also increases; hence the increase in visible turbidity

Turbidimetric measurement

T/F: Turbidimetric measurement can also be used for multicellular microbes

FALSE

Turbidimetric measurement cannot be used for multicellular microbes because there may be variability in proportionality

Turbidimetric measurement is recommended for _ cultures because bacteria of different sizes will also have different abilities to scatter light

pure

Explain turbidimetric measurement procedures

• From an isolated bacterial colony,

• you create a pre-culture (broth), then

• create a culture (broth) with standardized inoculum (from pre-cultured broth)

• Incubate at an appropriate temperature and O2 concentration

• Optical density (OD) measurement

• Plot standard and growth curves

T/F: Different colors of inoculum in spiral plating may indicate different dilutions

TRUE

T/F: Unless you’ve inoculated log-stage bacteria, turbidity measurement will always measure the total count

TRUE

Stationary, decline = total count because there’s equal division and death rate in stationary (UNLESS CELL LYSES)

T/F: If cells die due to lysis in stationary phase, turbidity indicates total count

FALSE

Turbidity = viable count at this point since spectro won’t be able to measure lysed cells; if cells just die = total count

T/F: It is reasonable to assume turbidity in log-stage bacterial inoculum as viable count

TRUE

Since log-stage bacteria behave identically, then if 1 is viable, most likely they’re all viable; turbidity = viable count

Standard curve vs. Growth Curve

• Standard curve (linear)

  • Optical density (Y) vs. Number of cells (X)

• Growth curve (logarithmic)

  • Optical density (Y) vs. Time (X)

Standard curve or Growth curve?

Optical density (y) vs. Time (x) = logarithmic = Growth curve

Standard curve or Growth curve?

Optical density (y) vs. Number of cells (x) = linear = Standard curve

Principle behind turbidimetric measurement

• Rapid, widely used approach for estimating cell numbers in a solution, relying on the principle that

• Light is scattered in proportion to the number of cells (unicellular)

• As no. of cells increases, so does the amount of light being scattered, resulting in increased visibility of turbidity

• _ measures the unscattered light that passes through a sample

• Measures 480 nm (blue), 540 nm (green), 600 (orange), 660 (red)

• The shorter the wavelengths, the more sensitive, but at high cell densities, it is recommended to use longer wavelengths (less sensitive)

Spectrophotometer

Why is it recommended to use longer wavelengths (for spectrophotometry) for high cell densities?

• Bc shorter wavelengths are more sensitive and could cause more light scattering, potentially leading to overestimation of OD

• Meanwhile, using longer wavelengths reduce sensitivity to light scattering, providing a more accurate measure of total biomass at high concentrations

_ is the most common wavelength to measure OD bacteria

600 nm (orange)

T/F: Optical density measures scattered light, while spectrophotometer measures unscattered light

TRUE

OD = scattered light = no. of cells

T/F: Optical density is a measure of viable count, not total count

FALSE

Optical density is a measure of total count. It does not distinguish viable from non-viable cells.

_ relates cell number (microscopic or viable count) to turbidity, but its proportionality only holds within limits

Standard curve

T/F: OD600 directly measures only viable cells in a bacterial culture

FALSE

The only 2 instances where turbidity definitely indicates viable count would be _

• When analyzing log-stage bacterial inoculum

• When dead cells in inoculum have undergone lysis

T/F: 480 nm is more sensitive than 660 nm for detecting bacterial growth

TRUE

T/F: OD600 is the best wavelength for bacterial growth monitoring because bacteria are orange in color

FALSE

OD600 is widely used because it provides moderate sensitivity and avoids extreme light scattering, but it has no relation to bacterial color.

The more cells, the more light scattered, the higher the OD, but the _ (for spectrophotometer)

less unscattered light measured by spectrophotometer

T/F: Optical density and light absorption are the same

FALSE

• Optical density measures cell density through light scattered by cells

• Light absorption = light absorbed by cells

Limitation of standard curve; how to resolve this issue

• It has the assumption that optical density = cell number, but there’s a limit to that

• This proportionality only holds within limits, e.g., only for unicellular cells or microbes that grow evenly distributed in suspension in liquid medium

• In this case, the proportionality only held within OD 0.5, so what you can do is dilute your sample if it exceeds OD 0.5, measure again, then multiply reading accordingly

  • e.g., If diluted 1:10 and OD = 0.6, actual OD 0.6 (10) = 6.0

  • Actual OD = OD measured x DF

Sample Problem on Optical Density and Dilution

A researcher is measuring the optical density (OD) of a bacterial culture at 600 nm. The spectrophotometer reads an OD of 1.2, but the lab protocol states that OD measurements above 0.5 are unreliable due to excessive light scattering.

To obtain an accurate reading, the researcher dilutes the sample 1:5 (1 part culture + 4 parts sterile medium) and measures the OD again. The spectrophotometer now reads 0.3.

Questions:

1. What is the actual OD of the undiluted bacterial culture?

2. If the OD is proportional to cell concentration, how much more concentrated is the original sample compared to the diluted one?

• Actual OD = OD measured (after exceeding OD limit) x DF

  • = 0.3 × 5 = 1.5

• Since OD is proportional to cell concentration, the original culture is 5 times more concentrated than the diluted one

Explain figure shown

• Growth curve = optical density (y) vs. time (x)

• This allows us to compare the growth rate of microorganisms

• Organism A grows faster than Organism B because it reaches a much higher OD at a shorter period of time (or less doubling time = faster doubling time)

• This implies that the slower-growing organism (B) would have a higher doubling time than faster-growing organism (A)

T/F: In a growth curve, a slower-growing organism would usually have higher doubling time than faster-growing organism

TRUE

T/F: Spectrophotometer measures the scattered light that passes through a sample

FALSE

Spectrophotometer measures the unscattered light that passes through a sample

Advantages and Disadvantages of Turbidimetric Measurement

• Advantages qrn

  • Quick and easy to perform

  • Readily repeatable

  • No adverse effects on cells (u can use it again as long as u keep it sterile)

• Disadvantages vluo

  • Very sensitive (can lead to changes in reading if u disrupt/agitate sample)

  • Limited to unicellular microbes, microbes that grow evenly distributed in suspension in liquid medium

  • Prone to underestimation

    • especially clumps/biofilms, which may contain hundreds of cells, but only scatter light once

    • <10⁷ cells/mL = not detected

  • Prone to overestimation

    • Deeply colored cultures

    • Suspended matter

• _ refers to the statistical determination of viable cells

• Suitable for samples with too few organisms or cells that do not grow on solid medium, e.g., agar

• Commonly used for food and water sanitation studies (coliform detection)

Most Probable Number (MPN)

3 indicators of a positive result in MPN tubes

get

1. Gas production (bubbles)

2. Effervescence

3. Turbidity

Explain MPN procedure

1. Homogenize sample

2. Inoculate 3 sets of tubes with different vols of inoculum, e.g., 10 mL, 1 mL, 0.1 mL (each responding to diff dilution)

3. Incubate

4. Score tubes as (+) or (-)

   1. (+) = both gas production, turbidity

5. If after 24 h, there no growth (or just turbidity but not gas) = negative

6. e.g., 10 mL = 5; 1 mL = 2, 0.1 mL = 0

   1. Could be 3-tube method, 5-tube method

   2. MPN index = 50 organisms / 100 mL

Comprehension check for MPN index

• Solve ff. table

• Sample A = 160 MPN/g

  • 40 - 420 MPN/g

• Sample B = 20 MPN/g

  • 4.5 - 42 MPN/g

Explain other way through which MPN may be performed

1. Create serial dilution of sample

2. 1 mL of each dilution is inoculated into triplicates of growth medium

3. Observe growth; 1st set of dilution tubes that fails to grow will be used to bracket a set of 3 dilutions (include 2 preceding dilution tubes)

4. Use MPN table, multiply MPN index to DF of middle tube set

   1. MPN index = 0.43 × 10^4 = 4.3×10³ microbes/mL of sample

Advantages and Disadvantages of MPN

• Advantages rs

  • Relatively simple and easy

  • Specific groups of organisms may be determined using appropriate selective and differential media

• Disadvantages lvop

  • Large volumes of glassware required

  • Lack of opportunity to observe colony morphology of organisms

  • Lack of precision (generally higher than SPC)

Culture-dependent technique

• Involves use of membrane filter with pore size that retains microbial cells but allows diluent or water to pass

• Bacteria = _ um

• Fungi = _ um

• Membrane filtration

• Bacteria = 0.22 um

• Fungi = 0.45 um

Explain membrane filtration procedure

1. Filter sample through membrane filter with pore size appropriate to microbe-of-interest

2. Place membrane to an agar plate or a medium saturated-absorbent pad

3. Incubate

4. Enumerate colonies

Culture-dependent technique

• Involves counting microcolonies in an agar-layered microscope slide

Microscope colony count

Explain microscope colony count procedure

1. Inoculate agar-layered microscope slide with sample

2. Incubate

3. Dry

4. Stain

5. Count microcolonies under microscope

Culture-dependent technique

• Viable count

• Faster than conventional plate count

• Food homogenate is diluted in tubes of molten agar

Agar Droplet Count

Explain agar droplet count procedure

1. + 1 mL of homogenate into (9 mL) of agar tube 1, mix

2. then 0.1 mL of agar tube 1 mix to 9.9 mL of agar tube 2, mix

3. then 0.1 mL of agar tube 2 mix to 9.9 mL of agar tube 3, mix

4. Use sterile capillary pipette (ideally delivering 0.033 mL/drop) to transfer five 0.1 mL droplets (per dilution) to bottom of petridish

5. Incubate

6. Enumerate colonies with 10x viewer

Miles and Misra vs. Agar Droplet Count

• Miles & Misra

  • Pre-solidified media

  • Only 1 replicate for each dilution

  • Diluent = liquid (e.g., Peptone water)

• Agar Droplet Count

  • Empty plate (no media)

  • Have several replicates for each dilution

  • Diluent = agar

Culture-dependent technique

• Estimates number of viable organisms

• Properly prepared supernatants of food are added to standard solutions of dye

• Time for dye reduction is inversely proportional to number of organisms

  • Longer amount of time it takes for dye to reduce = smaller no. of organisms

  • Faster/shorter amount of time for dye reduction = larger no. of organisms

• Methylene blue = blue → white

• Resazurin = blue → pink

Dye reduction

Advantages and disadvantages of dye reduction

• Advantages siro

  • Simple

  • Inexpensive

  • Rapid

  • Only viable cells actively reduce dyes

• Disadvantages

  • Not all organisms reduce dyes equally

  • Not applicable to food specimens containing reductive enzymes

Culture-dependent technique

• Excellent method for enumerating fastidious anaerobes and microaerophiles

• Predetermined amounts of molten inoculated agar are added to tube

• Agar is made to solidify as a thin layer on the inside of the tube

• H2 + CO2 (80:20) gases to make it anaerobic

Roll tube

Culture-dependent technique

• Consists of 2 plastic films

  • Attached together on one side

  • Consists of culture medium ingredients

  • Consists of cold-water-soluble jelling agent

• Correlated with conventional plate method

Rehydratable dry film

Culture-dependent technique

• Measures enzyme activity through fluorescence

• Enzymes + substrate → fluorescent compounds

• Fluorescence is detected under long-wave UV light

SimPlate (for HPC)

8 different types of microbiological examination of surfaces

ssr adsus

• Swab

• Swab-rinse

• Replicate organism direct agar contact (RODAC)

• Agar syringe / “Agar sausage”

• Direct surface agar plating

• Sticky film

• Ultrasonic

• Spray gun

_ is the oldest and most widely used method for the microbiological examination of surface

Swab method

• _ involves use of cotton or calcium alginate swab

• Templates may be prepared with openings corresponding to size of area to be swabbed

• Exposed swab is suspended in suitable diluent

• Can be stored at refrigerator temperatures until plated

Swab-rinse method

• _ involves raised agar surface in special Petri plates

• When plates are inverted, hardened agar makes direct contact with surface

Replicate organism direct agar contact (RODAC)

• _ creates a hollow cylinder out of a 100-mL syringe (remove needle end)

• Layer of agar is pushed beyond end of barrel by means of plunger and pressed against surface to be examined

• Exposed layer is cut off and placed in petri-dish, followed by incubation and colony enumeration

• Agar sausage = employ plastic tubing rather than modified syringe

Agar syringe method

• _ involves pouring molten agar directly onto surface or utensils to be assessed

• Upon hardening, agar mold is placed in Petri dish and incubated

• Enumeration of particles containing viable microorganisms

Direct surface agar plating

• _ involves pressing sticky film or tape against surface to be examine

• Press exposed side on agar plate

• Less effective than swabs in recovering bacteria from wooden surfaces

Sticky film

• _ is used for small and removable surfaces

• Immersed in a diluent

• Energy generated effects the release of microorganisms into diluent

Ultrasonic devices

• _ is based on impingement of a spray of washing solution against a circumscribed area of surface

• Subsequent plating of washing solution

• Much more effective than swab method in removing bacteria from meat surfaces

Spray gun

Major characteristics of a metabolically injured microorganism

iind

• Increased lag phase (wait it out) and inability to form colonies on selective media

• Increased sensitivity to various selective agents

• Nonfunctional ribosomes

• Damaged DNA and cell membrane

Enumerate environmental stressors causing metabolic injury

ad sfd iass heo

• Antibiotics

• Dyes

• Sublethal heat

• Freezing and freezedrying

• Drying

• Irradiation

• Aerosolization

• Sodium azide

• Salts

• Heavy metals

• Essential oils

• Other chemicals

How do you conduct detection and recovery for metabolically injured microorganisms?

• Plate aliquots separately into a general purpose / nonselective medium and a selective medium

• Difference between number of colonies on 2 media is a measure of the number of injured cells in the original culture or population

• Colonies that grew on general purpose and did not on selective are most likely the MI ones, so inoculate these onto a recovery medium, e.g., nutrient broth

Explain advantages and disadvantages of culture-dependent techniques

• Advantages arqh

  • Assesses living + culturable microbes

  • Allows us to recognize viable cells

  • Easy to quantitate cells in sample

  • High sensitivity with appropriate media

• Disadvantages rhtp

  • Risk of contamination

  • High skill level is necessary for optimal results

  • Time- and resource-intensive

  • Relies on phenotypic biochemical characterization