microbio 304 - lab exam #1

[LO] Common laboratory hazards

Biological hazards (microbes and cultures), chemical hazards (toxic, corrosive, irritating, or flammable substances), and physical hazards (open flames, hot equipment, broken glass, and sharp objects).

[LO] Basic laboratory safety

Wash hands, wear safety glasses, disinfect the bench, organize supplies, follow instructor directions, and dispose of materials in designated containers.

[LO] Proper laboratory disposal

Dispose of biological, chemical, glass, and regular waste according to the laboratory's specific instructions. Do not assume all waste goes into the same bin.

[LO] Handling plates that may contain mold

Do not open plates. Inspect through the lid or bottom. Parafilm plates with fuzzy or fluffy colonies that may be mold and discard them in the designated bin as instructed.

[LO] Concentration

The amount of solute dissolved in a given volume of solution.

[LO] Dilution

Reducing a solution's concentration by adding solvent.

[LO] C1V1 = C2V2

C1 = initial concentration; V1 = volume of stock needed; C2 = desired final concentration; V2 = final total volume. Use consistent concentration and volume units.

[LO] Solving for stock volume using C1V1 = C2V2

V1 = (C2 × V2) / C1.

[LO] Calculating solvent volume

Solvent volume = desired final volume − stock volume.

[LO] C1V1 = C2V2 example

To make 10 mL of 2 mg/mL solution from 10 mg/mL stock, use 2 mL stock and add solvent to a final volume of 10 mL (8 mL solvent).

[LO] Molarity (M)

Molarity = moles of solute / liters of final solution. Units are mol/L.

[LO] Calculating moles

Moles = molarity × volume in liters.

[LO] Preparing a molar solution from a solid

Mass (g) = desired molarity (mol/L) × final volume (L) × molecular weight (g/mol). Dissolve the solid and bring the solution to the desired final volume.

[LO] Percentage solution: % (w/v)

Grams of solute per 100 mL of final solution. For example, 5% (w/v) = 5 g per 100 mL final solution.

[LO] Percentage solution: % (v/v)

Milliliters of liquid solute per 100 mL of final solution. For example, 10% (v/v) = 10 mL per 100 mL final solution.

[LO] Percentage solution: % (w/w)

Grams of solute per 100 g of total solution. For example, 5% (w/w) = 5 g per 100 g final solution.

[LO] Dilution factor

Dilution factor = final total volume / original sample volume.

[LO] A 1:10 dilution

One part original sample in ten parts total solution. For example, 1 mL sample plus 9 mL diluent produces a 10-fold dilution.

[LO] Serial dilution

A series of successive dilutions in which each diluted sample is used to prepare the next dilution.

[LO] Overall serial dilution factor

Multiply the individual dilution factors. Three successive 1:10 dilutions produce an overall 1:1,000 dilution.

[LO] Concentration after dilution

Final concentration = original concentration / dilution factor.

[LO] Micropipette

A precision instrument used to measure and transfer small volumes of liquid, usually in microliters (µL).

[LO] Choosing a micropipette

Choose a pipette whose specified volume range includes the desired volume. Never set it outside its allowed range.

[LO] Setting a micropipette

Adjust the volume dial and check the display before pipetting.

[LO] Micropipette first stop

Used when aspirating the set volume and when beginning to dispense the measured liquid.

[LO] Micropipette second stop

Used to push out the remaining liquid during dispensing.

[LO] Aspirating liquid with a micropipette

Attach a clean tip, press the plunger to the first stop, immerse the tip slightly in the liquid, and slowly release the plunger while holding the pipette upright.

[LO] Dispensing liquid with a micropipette

Place the tip against the inside wall of the receiving container, depress the plunger to the first stop and then the second stop, and withdraw the tip while keeping the plunger depressed.

[LO] Preventing pipetting errors

Use the correct pipette and tip, set the correct volume, aspirate slowly, avoid bubbles, and change tips between different samples.

[LO] Accuracy

How close a measurement is to the true or accepted value.

[LO] Precision

How close repeated measurements are to one another.

[LO] Spectrophotometer

An instrument that measures how much light a sample absorbs at a selected wavelength.

[LO] Absorbance

A measure of how much light a sample absorbs.

[LO] Wavelength

The wavelength determines the light used to measure the sample. The appropriate wavelength depends on the substance being measured.

[LO] Spectrophotometer blank

A reference containing the appropriate solvent or medium without the substance being measured.

[LO] Purpose of blanking a spectrophotometer

To establish a baseline and account for background absorbance from the solvent, medium, and cuvette.

[LO] Proper cuvette handling

Fill appropriately, remove bubbles, wipe the outside clean, avoid fingerprints in the light path, and maintain consistent orientation.

[LO] Beer-Lambert law

A = εlc, where A = absorbance, ε = molar absorptivity, l = path length in cm, and c = concentration in mol/L when using molar absorptivity.

[LO] Calculating concentration using Beer’s law

c = A / (εl).

[LO] Calculating absorbance using Beer’s law

A = εlc.

[LO] Relationship between absorbance and concentration

Under suitable Beer-Lambert law conditions, absorbance is directly proportional to concentration when wavelength and path length remain constant.

[LO] Using absorbance to determine an unknown concentration

Measure absorbance after blanking the spectrophotometer, then use Beer’s law or a calibration curve to estimate concentration.

[LO] Calibration curve

A graph relating known concentrations to measured absorbance, used to estimate the concentration of an unknown sample.

[LO] Why Beer’s law may not be linear at all concentrations

Very concentrated samples, background interference, unsuitable measurement conditions, or instrument limitations can cause deviations from linearity.

[LO] OD600

Optical density measured at a wavelength of 600 nm; used to estimate microbial growth in a liquid culture.

[LO] Purpose of measuring OD600

To estimate the concentration of suspended microbial cells without counting every cell individually.

[LO] How OD600 estimates cell concentration

Cells scatter light, reducing the amount of light reaching the detector. Greater turbidity generally produces a higher OD600.

[LO] High OD600

Generally indicates a more turbid culture with more suspended cells, provided the reading is within the useful measurement range.

[LO] Low OD600

Generally indicates a less turbid culture with fewer suspended cells.

[LO] Does OD600 measure living cells only?

No. OD600 measures turbidity from suspended particles and does not distinguish living cells from dead cells.

[LO] Why dilute a culture before measuring OD600

To bring a highly concentrated culture into a more reliable measurement range.

[LO] OD600 reporting range specified in the PPT

Future lab activities should report OD600 readings between 0 and 1.0. The initial practice activity is an exception that allows readings above 1.0.

[LO] Adjusted OD600

The estimated OD600 of the original, undiluted culture after correcting for the dilution.

[LO] Adjusted OD600 equation

Adjusted OD600 = measured OD600 × dilution factor.

[LO] Adjusted OD600 example

A 1:10 dilution gives a measured OD600 of 0.25. Adjusted OD600 = 0.25 × 10 = 2.5.

[LO] What OD600 should be reported after dilution?

Report the adjusted OD600, not just the measured reading of the diluted sample.

[LO] Why blank with the appropriate medium?

To account for background absorbance or scattering from the medium and better isolate the culture's signal.

[LO] Does OD600 give an exact cell count?

No. OD600 estimates cell concentration. Converting it to cells/mL requires an appropriate calibration or established relationship for that organism and setup.

[PPT] Aseptic technique

Procedures used to prevent contamination of cultures, materials, equipment, and the surrounding environment.

[PPT] Why aseptic technique matters

It prevents unwanted microorganisms from contaminating cultures and helps prevent microbes from escaping into the environment.

[PPT] Pure culture

A culture containing a single microbial species, isolated from a mixed population.

[PPT] Why obtain a pure culture?

Most environments contain multiple microbial species. A pure culture allows one species to be studied without interference from other microorganisms.

[PPT] Streak plate method

A technique that separates microbial cells across an agar plate so that isolated colonies can develop.

[PPT] Sterilizing inoculating loops and needles

The PPT describes using a Bunsen burner or incinerator to sterilize loops and needles.

[PPT] Sterilizing growth media

The PPT identifies autoclaving and filtration as methods for sterilizing media, as appropriate.

[PPT] Lab arrival routine

Wash hands, put on safety glasses, disinfect the bench, gather supplies, and document results from the previous class.

[PPT] Observation

Raw data collected during an experiment, such as six colonies on a thumbprint plate.

[PPT] Interpretation

An explanation or meaning assigned to experimental observations.

[PPT] Does no growth mean no microbes were present?

No. The microbes may not grow under the conditions provided. No single medium supports the growth of every microorganism.

[PPT] Can you calculate the percentage of all thumb microbes that grew?

Not from the thumbprint experiment described in the PPT because the total number of microbes originally present on the thumb is unknown.

[PPT] Specific and predictive hypothesis

A hypothesis identifying the predicted change, experimental conditions, variable being tested, and how the outcome will be measured.

[PPT] Components of a good experimental prediction

Predicted increase or decrease, outcome measured, growth conditions, active ingredient and its concentration, and measurement method.

[PPT] Liquid culture

Microorganisms grown in a liquid growth medium inside a tube or other container.

[PPT] Cloudy liquid culture

Cloudiness generally indicates microbial growth because suspended cells make the medium turbid.

[PPT] Goal of Lab Activity 2.2

Practice transferring microbes safely without allowing cultures to escape or become contaminated.

[PPT] Tube positioning during transfer

Hold the source and recipient tubes in the same hand, with their caps or plugs loosened but not yet removed.

[PPT] When to flame an inoculating loop or needle

Flame it before obtaining the inoculum and again after completing the transfer, following the demonstrated procedure.

[PPT] Why cool a loop or needle after flaming?

To avoid damaging microorganisms when collecting inoculum and to prevent hot metal from causing splattering.

[PPT] Flaming tube mouths

During the demonstrated transfer procedure, lightly flame the tube mouths and replace the caps or stoppers.

[PPT] How to flame a loop or needle

Move it slowly through the flame from the base of the wire toward the tip to avoid spattering.

[PPT] Tube labeling

Write on the glass using the provided marker. Do not label caps or hold tubes by their caps.

[PPT] Incubating liquid cultures

Keep tubes containing liquid in the designated beaker in the basket or tray. Do not lay tubes sideways or lean them against the incubation bin.

[PPT] Incubation temperature for Lab Activity 2.2

30°C.

[PPT] Organisms used in Lab Activity 2.2

The PPT specifies E. coli and Serratia marcescens for the different tubes in the tube-transfer activity.

[PPT] Spectrophotometer interface for absorbance

Select “SPEC 200E Modern Interface” from the Home Menu and press Enter.

[PPT] Setting the absorbance wavelength

Navigate to Measurement, select the appropriate wavelength using the arrows or dial, and press Go.

[PPT] Spectrophotometer setting for cell growth

Select the OD600 setting from the Home Menu.

[PPT] Before taking absorbance or OD600 readings

Blank the spectrophotometer correctly before measuring samples.

[PPT] Preparing cell-free supernatant in Lab Activity 1.5

Transfer 1.3 mL of culture into a microcentrifuge tube, centrifuge for one minute at maximum speed using a counterweight, and measure 1 mL of cell-free supernatant in a cuvette.

[PPT] Cell-free supernatant

The liquid remaining after cells have been separated from a culture, in this activity by centrifugation.

[PPT] Expected OD600 of cell-free supernatant

Approximately 0.0 because most cells have been removed; residual particles may produce a nonzero reading.

[PPT] Purpose of a centrifuge counterweight

To balance the rotor and help prevent unsafe vibration or damage during centrifugation.

[PPT] Goal of the dilution proficiency activity

Prepare a dilution of a pink stock solution, measure its absorbance, and calculate the concentrations of the diluted sample and original stock.

[PPT] Dilution proficiency volume limit

Maximum volume of 2 mL; use one cuvette per person.

[PPT] Reporting OD600 readings above 1.0

The initial practice activity allows readings above 1.0 for familiarization. Future lab activities should report readings between 0 and 1.0 after diluting as needed and calculating adjusted OD600.

[LO] Percentage equation (% w/v)

% (w/v) = (grams of solute / mL of final solution) × 100. Used for solid solute dissolved in liquid.

[LO] Percentage equation (% v/v)

% (v/v) = (mL of solute / mL of final solution) × 100. Used for mixing liquids.

[LO] Percentage equation (% w/w)

% (w/w) = (grams of solute / grams of final solution) × 100. Used when measuring solute and total solution by mass.

[LO] Calculate grams for a % (w/v) solution

Grams of solute = (% × final volume in mL) / 100.

[LO] Calculate mL for a % (v/v) solution

mL of solute = (% × final volume in mL) / 100.