Sample Collection Methods for MicrobiologyPreparations, Equipment, Collection, QA/QC

Important Concepts

What special considerations are there when preparing for and collecting microbiology samples?How are samples collected (bacteria, coliphage, protozoa, viruses)?Where can I find more information on microbiology sampling?

Refer to USGS National Field Manual

Section 7.1 Fecal Indicator Bacteria7.1.1 – 7.1.2: sample collection information

Section 7.2 Fecal Indicator Viruses7.2.1 – 7.2.2: sample collection information

Section 7.3 Protozoa Pathogens7.3.1 – 7.3.2: sample collection information

http://water.usgs.gov/owq/FieldManual/Chapter7/index.htmlMore Resources: USGS Ohio Water Microbiology Laboratory

Cleaning and Disinfecting equipment

Wash equipmentUse dilute liquinox, rinse with tap water and deionized water 3-5 times

Autoclave equipmentWrap equipment in Kraft paper, autoclavable bags, or aluminum foil

Sterilize non-autoclavable equipmentChemical disinfection

Types of Sterilization

Steam (autoclave)Dry heat (seldom used)Filtration ChemicalIonizing radiation (ultraviolet light)

Autoclaving

Glassware and otherdry materials—15 min.Contaminated and discarded materials—30 min.Liquids, total volumes

Up to 250 mL—15 min.500 to 1,000 mL—30 min.> 2,000 mL—45 min. or more

Chemical disinfection: Indicator bacteria & coliphage

50 to 70% ethanolfor sterilizing spigots

50 to 70% isopropyl alcoholfor working surfaces

50 mg/L sodium hypochlorite (0.005%)Prepare by adding 1 mL of household bleach to 900 mL water – bring volume up to 1,000 mL Soak non-autoclavable equipment for 30 min.Neutralize with sterile sodium thiosulfate for 5 min. Rinse with (or circulate) 0.5 mL of 10% solution sodium thiosulfate per 1 L sterile water (0.005%)Rinse with sterile deionized water

Chemical disinfection: enteric viruses

Alcohol is not effectiveUse 1,000 mg/L sodium hypochlorite (0.1%)

Prepare by adding 19 mL of household bleach to 900 mL water – bring volume up to 1,000 mL (most household bleach is about 5% sodium hypochlorite).Adjust pH of the solution to neutral (pH 7) or below.Soak non-autoclavable equipment for 30 min.Neutralize with sterile sodium thiosulfate for 5 min. Rinse with (or circulate) 0.5 mL of 10% solution sodium thiosulfate per 1 L sterile water (0.005%)Rinse with sterile deionized water

Chemical disinfection: Cryptosporidium and Giardia

Autoclaving is ineffectiveDoes not destroy epitopes. These proteins on the surface of cells react with antibodies to produce a positive test result.

120 g/L sodium hypochlorite (12%)Soak equipment for 30 min.Prepare by adding full strength pool bleach.To neutralize, rinse 3 X with sterile deionized water only.

Chemical disinfection: Protozoa

Alternative to using 12% hypochloriteClean equipment

Soak in dilute liquinox for 30 minutesScrub wellRinse 3-5 times with tap and DIW

Autoclave equipmentAdd extra QC samples

Ultraviolet radiation

The 254 nm range is used for germicidal purposes Must penetrate to surface to be effectiveVegetative microorganisms are more susceptible than bacterial spores

Amendments (add after cleaning)

Sodium thiosulfate Use if sample water may contain residual chlorine or other halogensAdd 0.5 mL of 10% sol’n. per 1 L sample (0.005%)

EDTA (ethylene diaminetetracetic acid)Use if sample water may contain trace metals (eg. copper, nickel, zinc) in concentrations > 10 mg/LAdd 0.3 mL of ~0.04% sol’n. per 100 mL sampletightly binds metal ions.

Preparations for membrane filtration

Keep poured plates on ice before use.Note expiration dates of agar.Sterile buffer needs to be kept on ice only after bottle is opened.PREHEAT INCUBATORS.

Sample collection: bacteria, coliphage, protozoa

Maintain sterile conditions, use aseptic technique even in fieldStore samples on ice until processingKeep samples out of sunlightAlways leave 2.5 - 5 cm of headspace in the sample bottle

GW sample collection: bacteria, coliphage, viruses

Follow appropriate well-purging techniques Wells with an in-place pump

For most wells, collect the sample directly from the tap.Remove screens, filters, etc.Flame sterilize tap with ethanol and rinse with sterile DI waterDo not sample treated water

GW sample collection: bacteria, coliphage, viruses

Wells without an in-place pumpUse a peristaltic or vacuum pump with autoclavable tubing (shallow wells), orUse a sterile bailerDisinfect the pump with

• Chlorine, or • Clean thoroughly with Liquinox

• Add extra QC samples!

SW sampling: bacteria, coliphage, protozoa

Microorganisms are not evenly or randomly distributed in water bodies Use depth- and width-integrating sampling methods for flowing streams (EWI or EDI)Small, shallow, or low-velocity streams or well-mixed cross-sections may be sampled using the hand-dip method

Sample at least 1 ft. below the water surface

SW sampling: bacteria, coliphage, protozoa

Lake, reservoir, estuary, and ocean samples should be collected with point samplersFor beach water, use the hand-dip method

Knee-depth water, or about 6 to 12 inches below the water surface

SW sampling equipment: bacteria, coliphage, protozoa

Use standard sampling equipment with autoclavable bottles and nozzlesFor compositing samples, use a sterile 3-L or larger bottle

Preparations: enteric viruses

Autoclave or sterilize filter Clean equipment

Dilute liquinox through the samplerRinse with deionized water

Sterilize sampling apparatusCirculate 0.1% sodium hypochlorite solution through sampler for 30 minutesRun 0.005% sodium thiosulfate through sampler for 5 minutesRun STERILE DI water through the system

Sample collection: enteric viruses

Wear sterile gloves When assembling and sterilizing samplerFilter apparatus is double bagged and kept on ice for shipment

VIRUS SAMPLING APPARATUS

PumpIntake linePre-filter, if necessaryPressure regulatorPressure gageFilterWater meterDischarge line

Sample collection: Protozoa

Collect multiple samples using EWI or EDIComposite subsamples into a sterile cubitainer

Holding Times

GW & SW bacteria samples 6 hrs

Drinking water bacteria samples 30 hrs

C. perfringens samples 24 hrs

Coliphage samples 48 hrs

Enteric virus samples 48 hrs

Cryptosporidium & Giardiasamples

96 hrs

QUALITY ASSURANCE

Procedures to control non-measurable components of a project

Work plansTraining plansWritten protocols of non-standard proceduresCalibration and maintenance of equipment

QA FOR EQUIPMENT

Check temperatures of incubators and refrigerators daily.Check thermometers against NIST certified thermometer biannuallyCheck operation of autoclave quarterly using spore indicators

Keep records of equipment checks and maintenance in log books

QA FOR MEDIA AND REAGENTS

Products prepared from dehydrated media

Test each batch for growth of target and nontarget colonies.

Ready-to-use media platesTest plates at beginning & middle of sampling periodTest when lot numbers change

More QA FOR MEDIA AND REAGENTS

Observe all ready-to-use media, reagents, and buffer water for discoloration, cloudiness, color change, or contamination.Additional QC might be necessary if preparing rinse buffers in-house.Record and document all QC procedures and results in field notes

QA FOR MEMBRANE FILTERS

Membrane filters should completely wet within 15 seconds of being placed on agar with no dry areasColonies should be of expected size and shape Grid lines should not bleed or restrict colony developmentMembranes containing large areas with no colony development are questionable

QUALITY CONTROL

Measurable components of bias and variability for techniques and activities in

the field and laboratory

Filter and procedure blanksField and equipment blanksReplicatesMatrix spikesReference cultures

QUALITY CONTROL DEFINITIONS

BIAS—systematic error in a method or caused by some problem of the measurement system. Bias is positive (from contamination) or negative (from interference, loss, or degradation)

VARIABILITY—the degree of variation in independent measurements as the result of repeated application of the measurement process

QC DEFINITIONS:Membrane filtration blanks

FILTER BLANK—Sterile buffered water filtered before each sample to test for contamination.

PROCEDURE BLANK—Sterile buffered water filtered after each 10th sample to test for completeness and sterility of the filtering process.

QC DEFINITIONS: Reference samplesPure cultures analyzed in the same

manner as the sampleEnsures the procedure is correctly performed and tests the integrity of the mediumPositive and negative control cultures

QUALITY CONTROL: No. of samples needed

The numbers and types of QC samples are determined by the project chief to address project objectivesThe following references are available:

USGS National Field manualUSGS Ohio Water Microbiology Lab web site

QC required for indicator bacteria

Filter and procedure blanksField blanks every 10-20 samples

use sterile buffered waterReference cultures

each batch or new lot of ready-to-use platesquarterly, for daily or weekly samplingmore often for unstable agar (e.g. MI) or when analyst is inexperienced

QC: Indicator bacteria—cont.

ReplicatesEvery 10-20 samplesFor GW, recommend a replicate for every sampleFor SW, recommend split sequential replicates

HIGH VARIABILITY IS OFTEN CAUSED BY INCOMPLETE MIXING!!!

QC FOR COLIPHAGE

Field blanks and replicatesFor SW, every 10-20 samplesFor GW, every 10-20 samples only if required by data-quality objectivesUse sterile water for field blanks

QC FOR COLIPHAGE —Cont. Matrix spikes

Determines any matrix interference on the analytical methodSpiked in the labFirst received from a water source and every 20th sample thereafterShould be collected more often if there is a wide range of water types in the study

QC for Cryptosporidium and Giardia

Recommend a higher frequency due to the wide range of recoveries by this method.Matrix spikes

Extra 10-L sample needed for spikingFirst 2 samples from a new water source; every 20th sample thereafter (Method 1623)

QC: Enteric viruses

Matrix spikesMeasures analytical bias5% of samples

QC: Enteric viruses—cont.

Equipment blankDone before sampling beginsSterile DI water processed through all stages of sample collection and handling in a controlled environment

Field blankLike equipment blank but done under actual field conditions5% of samples>5% if contamination is found

Some quick thoughts on QA/QC procedures

Always test the sterility of filter apparatus, buffer, and utensils before processing the sampleDo not overload the autoclave and check the time charts for large volumes of liquidsObserve and be aware of expiration dates on perishables Refrigerate and process samples for indicator bacteria within 6 hours