plating one ml of the liquid would result in the number of cfu on the plate
to be equal to the number spores per one gram of waste.]

a)

b)

°)

B. Test Run

Triplicate plate 0.1 ml from the 10 ml concentrate in Step 8 above;

this dilution represents Plate A. [This step equates to a total
dilution of 1:10.]

Add 1.0 ml of the 10 ml concentrate in Step 8 above to 9.0 mls of
buffer solution (this represents a 1:10 serial dilution and is
fepresented as Dilution Tube B). Triplicate plate 0.1 ml of
Dilution Tube B; this dilution represents Plate B. [This step
equates to a total dilution of 1:100.]

Add 1.0 ml of Dilution Tube B above to 9.0 mls of buffer solution
(This represents an additional 1:10 serial dilution and is represented
as Dilution Tube C). Triplicate plate 0.1 ml of Dilution Tube C;

this dilution represents Plate C. [This step equates to a total
dilution of 1:1000).

Add 1.0 ml of Dilution Tube C above to 9.0 mls of buffer solution
(This represents an additional 1:10 serial dilution and is represented
as Dilution Tube D). Triplicate plate 0.1 ml of Dilution Tube D;
this dilution represents Plate D. [This step equates to a total
dilution of 1:10,000).

1. Follow protocols in II A. except run the treatment unit with specified
chemical inactivation agent concentrations.

2 Upon washing the membrane filter in Step 11.8 with 10 mls of buffer:

a)

b)

Triplicate plate 1 ml of buffer in Step 2 above via the pour plate
method (i.e., 1 ml of spore concentrate into 10-12 mls of liquid
agar. Vortex and pour into plate; this represents Plate A’. [This
step equates to no dilution factor, i.e., this number represents the
number of spores per gram of waste.]

Triplicate plate 0.1 ml of buffer in Step 2 above via the pour plate
method (i.e., 0.1 ml of spore concentrate into 10-12 mls of liquid
agar. Vortex and pour into plate; this represents Plate B'. [This
step equates to a 1:10 dilution factor.)

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Collect ten one (1) gram samples during the duration of the run (ie.,
collect samples at the beginning of waste discharge through final
discharge). NOTE: The amount, number and collection frequency of the
sample collection will be determined previously by trial runs. The
important consideration for this determination is to ensure that during the
span of the run, the test data collected provide an accurate reflection of the
level of microbial inactivation for the entire load.

Place the 1-gram samples immediately upon collection into pre-weighed
(combination weight of both liquid and tube) plastic screw cap tubes
containing an appropriate neutralizing solution and vortex vigorously for
5 minutes. NOTE: This step is required to neutralize chemical agent
activate at the time the waste exits the unit and is necessary to determine
actual microbial inactivation the treatment process and minimize the
inclusion of residual chemical activity that might be present. The amount,
concentration, and exposure time of the selected neutralizing agent must
be pre-determined so as to neutralize the specific chemical agent without
inhibiting growth of the biological indicator. Collection tubes are
pre-weighed, including neutralizing agent, to determine the weight of the
actual waste sample collected.

Construct an approximate 10-gram composite sample from the 10
tepresentative samples collected in Step 3. [This step provides for the
evaluation of the level of microbial inactivation of the entire load without
assaying each individual sample taken above.]

Decant, sieve, and filter as required to separate solid waste material from
the neutralizing liquid. Save liquid effluent. [This step is required to
wash bacterial spores from the collected waste sample. Protocols involved
in this rinsing step will be determined by trial runs to ascertain the best
mechanisms to adequately rinse and separate the solid waste components
from the liquid rinse.]

Wash and vortex solid materials a second time with neutralizing buffer.
Decant, sieve, and filter as required to separate solid waste material from
liquid. Combine liquid effluent with that obtained in Step 6. [This step
provides an extra wash to collect from the waste as many of the spores as
possible.]

Filter liquid through MilliporeTM filtration unit or equivalent to concentrate
retrieved spores on membrane filter. Wash filter with 10 mls of citrate or
other appropriate buffer. [This step concentrates retrieved spores to equa!
the number of spores from 10 grams waste/10 mls buffer or by factoring,
the number of spores from 1 gram waste per 1 ml buffer. For exampl>.

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nee


c) Add 1.0 mi of the buffer in Step 2 above to 9.0 mls of buffer
solution [this represents a 1:10 serial dilution and is represented as
Dilution Tube C'}. Triplicate plate 0.1 ml of Dilution Tube C';
this dilution represents Plate C'. [This step equates to a tota!

dilution of 1:100.}

Calculations

Using the equations found in Section C3 of "State Guideline for Approval of Alternate
Medical Waste Technologies", the following calculations are performed:

A Calculate initial imoculum in spores per gram waste.

1. 2 x 10” spores/15 Ibs. waste =

2 x 10” spores/6.8 x 10° grams waste =

3 x 10° spores/gram waste = inoculum = IC

IC=3x 10

B Calculate number of spores recovered.

1. Step One "Control" Data:

a b
Plate A - TMTC* TMTC
Plate B- TMTC TMTC
PlateC - TMTC TMTC
Plate D - 200 cfu** 210 cfu
*Too Many To Count
**Colony Forming Units

10

TMTC
TMTC

190 cfu

Accounting for the dilution factor of 10,000 for Plate D, the average recovery 9:
viable "Control" spores per gram equals 200 x 10,000 or 2,000,000 spores/gram

or 2 x 10° spores/gram.

RC =2x 10°

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APPENDIX C

EXAMPLE:
MICROBIAL INACTIVATION TESTING PROTOCOL FOR A
GRINDER/CHEMICAL MEDICAL WASTE INACTIVATION PROCESS


Step Two "Test" Results:

a b ¢
Plate A'- 50 cfu 48 cfu 52 cfu
Plate B'- 5 cfu 4 cfu 6 cfu
Plate C'- 1 cfu 0 cfu 0 cfu

The average recovery of viable "Test" Spores per gram equials 50 spores
per gram (no dilution factor).

RT=5x 10!

GQ Calculate Log,, Reduction.

1.

Step One "Control" Results:

LogipRC = Log,IC - Log,gNR; where
LogiRC = Log,o(2 x 10° spores/gram) = 6.301
LogylC = Log,o(3 x 10° spores/gram) = 6.477
LogyNR = LogyIC - Log,RC

LogyNR = 6.477 - 6.301 = 0.176

Log, NR = 0.176

Step Two "Test" Results and Log, Kill Calculation:

a) LogyKill = Log, IT - Log,pNR - Log, RT, where:
LogyIT = Log,IC = 6.477
LogioNR = 0.176
LogioRT = Log,o(5 x 10") = 1.699
b) Log), Reduction (Log, Kil), where:
LogyKill = 6.477 - 0.176 - 1.699 = 4.602

Log, Kill = 4.602

C-7


PREFACE

The following protocol is provided as an example of the steps and procedures required to
determine the level of microbial inactivation of a system that cannot ensure or provide integrity
of the biological indicator carrier (i.¢., test strip, ampule) through the treatment process to
recovery. This protocol is not intended to be all inclusive or meet all the variables or constraints
associated with the multiplicity of medical waste treatment technologies. However, the protocol
includes the components and the processes that require consideration to ensure the data recovered
and numeric calculations made accurately represent the true microbial inactivation level of the
treatment process.

This example provides a protocol for a chemical inactivation/ grinding medical waste treatment
process that does not allow the retrieval of the biological indicator carrier. For each step in the
protocol, an explanation or note is offered (in brackets) to provide rationale or background for
the step or process described. For the protocol provided, adherence to good microbial and
laboratory practices is essential for researcher and equipment operator safety and for the
generation of accurate data.

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EXAMPLE:
MICROBIAL INACTIVATION TESTING PROTOCOL FOR A
GRINDER/CHEMICAL MEDICAL WASTE TREATMENT PROCESS

L Materials

A Bacillus stearothermophilus spores as a suspension of 2 x 10'° initial inoculum.
NOTE: B. stearothermophilus spores were chosen as the spore of choice due to
the thermophilic nature of B. stearothermophilus and its ability to optimally grow
at elevated temperatures. Culturing collected waste samples at 60°C using B.
stearothermophilus spores as a biological indicator reduces the number of potential
cross contaminants that might arise on a culture plate. A spore suspension of 2
x 10" initial inoculum was chosen to provide an adequate number of recoverable
spores for determining a 4 Log), reduction. Determination of this concentration
may require trial runs to ascertain the recovery concentrations.

B Surrogate waste load constructed to contain by weight: 5% organic material and
95% plastics, cellulose, and glass. Total weight of sample to be between 15 and
20 pounds. NOTE: The surrogate waste load used in this example was
constructed to represent the typical medical waste composition that would be
treated by this system at the user site location. Surrogate waste loads may also
be constructed to replicate medical waste loads which challenge the efficacy of the
system. The sample weight of the load was selected as being representative of the
feed rate and typical loading conditions of the unit. Weight loads should be
constructed to mimic conditions of actual use.

IL. Protocols

A Control Run

1. Add 2 x 10" B. stearothermophilus spore suspension to surrogate waste
load. The spore suspension should be added as to not expose the
researcher or equipment operator to the biological indicator. To minimize
potential exposures and to adequately disperse the spore suspension
throughout the load, the spore suspension could be transferred into four or
more separate plastic screw-capped tubes. These tubes could subsequently
be equally dispersed throughout the surrogate waste load.

2 Load inoculated surrogate waste into the previously cleaned
(decontaminated) treatment unit and run unit without chemical inactivation
agent. [The unit should be previously decontaminated to minimize cross
contamination from spores originating from previous efficacy testing.]

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