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[ Microbiology Topics.Scott Sutton

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Measurement of MicrobialCells by Optical DensityScott Sutton

"Microbiology Topics" discusses various topics inmicrobiology of practical use in validation andcompliance. We intend this column to be a usefulresource fo r daily work applications.Reader comments, questions, and suggestionsare needed to help us fulfill our objective fo r thiscolumn. Please send your comments and sugges-tions to column coordinator Scott Sutton at scott.sutton@microbiol.org or journal coordinating edi-to r Susan Haigney at shaigney@advanstar.com.KEY POINTSThe following key poin ts are discussed:

Quality control (QC) microbiology tests requirecontrolled levels of inocula and require freshpreparations of cells for those inocula

The concentration of cells in a suspension canbe estimated by optical density, bu t this must beconfirmed by plate count

The optical density readings against cell mass arespecific to the microorganism species

The qualification of hese readings must be confirmed after major maintenance to the benchtop spectrophotometer (e.g., after replacementof th e bulb).

DETERMINATION OF INOCULUM FORTHEAETThe compendia) antimicrobial efficacy test (AET)requires inoculation of the product with microorganisms to a final concentration of approximately 106CFUfmL. Although this seems to be a minor point,it does serve to illustrate some of the inherent difficulties in microbiological testing and the need for

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experienced and academically trained microbiologiststo head th e laboratory.

The Pharmacopeia Europe (1) instruction on preparing the inoculum for th e AET states:

"To harvest the . . . cultures, use a sterilesuspending fluid ... Add sufficient suspendingfluid to reduce the microbial count to about lOSmicro-organisms per milliliter ..Remove immediately a suitable sample from each suspension anddetermine the number of colony-forming unitsper milliliter in each suspension by plate count ormembrane filtration (2.6.12). This value servesto determine the inoculum and the baseline touse in the test. The suspensions shall be usedimmediately."

There are, ofcourse, two problems with these instructions. The first is that the technician is instructed to usean inoculum of about 108 microorganisms per milliliterand then instructed to determine this by plate count.Colony forming units (CFU) and cells (micro-organismsand spores) are different measures. This will inevitablylead to difficulties as the unfortunate lab worker cannotguarantee the number ofcells in the suspension, onlythe number of CFU found. However, we can accept thescientific inaccuracy, as the numbers will generally workout. The more serious problem is the instruction to usethe plate count CFU for determination of he inoculumfor the test, and that t he suspension shall be used immediately. This quite frankly cannot be done. Ifyou use thesuspension immediately, the plate counts are unavailable;ifyou use the plate counts to set the inoculum, then thesuspension is at least a day old.

Contrast these instructions with those in the UnitedStates Pharmacopeia (USP) (2) for th e same exercise:

Scott Sutton, Ph.D., is owner and operator of The Microbiology Network (www.microbiol.org),which provides services to microbiology-related user's groups. Dr. Sutton may be reached by e-mail atscott.sutton@microbiol.org .

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"To harvest the .. . cultures, use sterile saline. Add sufficient ... to obtain a microbial countofabout 1 x 10" cfu per mL. ..{Note: The estimateof noculum concentration may be performed byturbidimetric measurements for the challengeorganisms. Refrigerate the suspension if it is notused within 2 hours].Determine the number of cfu per mL in eachsuspension ... o confirm the initial cfu per mLestimate. This value serves to calibrate the sizeof he inoculum used in the test."These USP instruct ions have the advantage of being

physically possible to perform, an advantage thatcannot be underrated. However, th e turbidometricmeasure of th e cells is also only an approximation ofCFU. Thus, th e instruction to confirm the numbers(after th e test is underway) with the plate count is animportant control on the test.

This article will explore the turbidometric approximation for cell numbers, the important controls onthe process, and the potential pitfalls to th e process.THEORYLight scattering techniques to monitor the concentration of pure cultures have the enormous advantagesof being rapid an d nondestructive. However, theydo not measure cell numbers nor do they measureCFU. Light scattering is most closely related to thedr y weight of the cells.Light is passed through th e suspension of microorganisms, an d all light that is not absorbed is reradiated. There is a significant amount of physicsinvolved in this, and those interested are referred tooptical treatises, particularly those discussing Buygens' Principle (a good choice is Light Scattering bySmall Particles by H C Van De Hulst). For our purposesit is enough to say that light passing through a suspension of microorganisms is scattered, and the amountof scatter is an indication of the biomass present inth e suspension. In visible light, this appears "milkywor "cloudyw to the eye (3). It follows from this thatif th e concentration of scattering particles becomeshigh, then multiple scattering events become possible.METHODSMcfarland Turbidity StandardsMcFarland standard s can be used to visually approximate the concentration of cells in a suspension. TheMcFarland Scale represents specific concentrationsofCFU/mL an d is designed to be used for estimatingconcentrations of gram negative bacteria such as E.coli. Note that this estimate becomes uncertain withorganisms outside th e normal usage as different species of bacteria differ in size and mass, as do yeast andgxpandjvt.com

Scott Sutton.Table: McFarland turbidity standardsMcFarland Scale CFU (x106/ml) 1% BaCij1% H2S04 (ml)0.5

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Microbiology Topics.

Figure: Spectrophotometer.

' IIightScattered Light I

DetectorSource Filter Slit MicroorganismSuspension

(although the relationship of absorption to CFU doesnot). However, in more concentrated suspensions thiscorrelation (absorption to dry weight) no longer holds.The linear range ofabsorption to estimated CFU is oflimited scope. For this reason, the calibration studymust demonstrate the linear range of the absorbancevs CFU values and the relevant values.ProcedureAs there are a variety of different instruments, therecannot be one single procedure. In general, the spectrophotometer can be set at a wavelength of 420-550nm. This wavelength must be standardized.

It is important to have the cells in known physiological state of growth. That is to say, as the cell sizevaries with phase of growth (i.e., lag, log, stationery),the approximate relationship between absorbance andCFU will also vary. A recommended practice might beto pass a single well-isolated colony twice on overnightcultures from the refrigerated stock, and harvest therapidly growing culture from the second passage. Thisalso will serve to minimize a source ofvariability forthe AET (6).

A second source of concern might be the cuvette usedfor the measurement-care must be taken to maintainth e correct orientation of the cuvette an d to protectit from damage that could affect the passage of light.Finally, it is necessary to blank the spectrophotometer(i.e., adjust th e absorbance reading to zero) using astandard, either water or the suspending fluid, an dmaintain this practice.CalibrationIt must be stressed that this calibration should be donefor all organisms. The size of he organism, any associated pigments, the preparation of he suspension, and

48 ]OURNAL OF VALIDATION TECHNOLOGY [WINTER 2011I

other factors all influence the readings. This calibration study should also be rechecked after changing thebulb on the light source, and should be reevaluatedthroughout the life of th e light bulb.

The calibration itself is simple to perform. Prepare aconcentrated solut ion of he organism, grown under theconditions that will be used for the test. Make a seriesofdilutions to cover the range of absorption measurements of interest; 5 to 8 dilutions are recommended.Immediately take the spectrophotometer readings insequence, and then take a confi rmatory readingof hefirst in series to confirm that no growth has occurred.The dilutions are then immediately plated for viablecount (serial dilution of he suspensions will be necessary). Graph the relationship between the absorbanceand th e CFU/mL after th e plate counts are availablean d use values in the linear range of his graph.

As there are several factors th at can affect this curve(e.g., quality oflamp output, size of slit, condition offilter, condition of detector, microorganism characteristic, etc.), this calibration should be confirmedwhenthe conditions of he assay change.CONCLUSIONSThe use of optical density to estimate CFU in a suspension is possible if basic precautions are taken. It isimportant to control the following: The physiological state of the organism The species of th e organisms The nature an d condition of the equipment.

Despite the inherent inaccuracy of the method, ifthe procedure is adequately controlled and calibrated,the estimation of microbial numbers by optical density (either by McFarland Standards or spectrophotometrically) is sufficiently accurate for use in preparing

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inocula for QC testing. The method offers the overwhelming advantages of being rapid, low cost, andnon-destructive.

Scott Sutton.

5. Koch, AL., "Thrbidity Measurements of Bacterial Cultures in Some Available Commercial Instruments, AnalBiochem 38:252-259, 1970.

6. Gilbert, P. eta!., "Inocula for Antimicrobial Sensitivity Testing: a Critical Review, J Antimicrob Chemother.REFERENCES 20:147-154, 1987. JVT

1. EP, s.1.3 Efficacy of Antimicrobial Preservation,"Pharm Eur 6.6 pp, 5129-5130. ARTICLE ACRONYM LISTING

2. USP, Antimicrobial Effectiveness Testing, UnitedStates Pharmacopeia, 2011.

3. Koch, AL., "Growth Measurement," Methods for Gen-eral and Molecular Bacteriology, Gerhardt, Pet a!. (ed)American Society for Microbiology, Washington, DC.p. 248-277, 1994.

4. Smibert, RM and NR Kreig. "Phenotypic Charac terization Section 25.4.9 Methods for General and MolecularBacteriology, Gerhardt, Pet a!. (ed) American Society forMicrobiology, Washington, DC. p. 607-654, 1994.

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AETCFUQCUSP

Antimicrobial Efficacy TestColony Forming Un itQuality ControlUnited States Pharmacopeia

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