508.8-44 test process

4.1.1 Test Chamber

Construct the chamber and accessories in a manner to prevent condensation from dripping on the test item. If required, filter-vent the chamber to the atmosphere to prevent the buildup of pressure and release of spores into the atmosphere.

4.1.2 Sensors

Determine the relative humidity by employing either solid state sensors whose calibration is not affected by water condensation, or by an equivalent method such as fast-reacting wet-bulb/dry-bulb sensors or dew point indicators. Sensors that are sensitive to condensation, such as the lithium chloride type, are not recommended for tests with high relative humidity levels. A data collection system, including an appropriate recording device(s), separate from the chamber controllers is necessary to measure test volume conditions. If charts are used, use charts readable to within ± 2 °C (± 3.6 °F). If the wet-wick control method is approved for use, clean the wet bulb and tank and install a new wick before each test and at least every 30 days. Ensure the wick is as thin as realistically possible to facilitate evaporation (approximately 1/16 of an inch thick) consistent with maintaining a wet surface around the sensor. Use water in wet-wick systems that are of the same quality as that used to produce the humidity. When physically possible, visually examine the water bottle, wick, sensor, and other components making up relative humidity measuring systems at least once every 24 hours during the test to ensure they are functioning as desired.

4.1.3 Air Velocity

Minimal airflow is recommended for this method. It has been found that velocities greater than 0.5 meters/second (98 feet/minute) retard the growth of fungus. If a wet bulb sensor is used, recommend an air velocity flowing across the wet bulb sensor of not less than 4.6 meters/second (900 feet/minute, or as otherwise specified in sensor response data), and ensure the wet wick is on the suction side of the fan to eliminate the effect of fan heat. Maintain the flow of air anywhere within the envelope of air surrounding the test item between 0.5 and 1.7 meters/second (98 to 335 feet/minute).

MIL-STD-810H

METHOD 508.8

4.1.4 Decontamination

Prior to testing, ensure the chamber is decontaminated in accordance with the guidance at Annex A.

4.2.1 Relative Humidity

In addition to the requirements appropriate for Method 507.6 Humidity, and water purity as described in Part One, paragraph 5.16, determine the relative humidity by employing either solid state sensors whose calibration is not affected by water condensation or by an approved equivalent method such as fast-reacting wet bulb/dry bulb sensors. Do not use lithium chloride sensors because of their sensitivity to condensation.

• a. When the wet bulb control method is used, clean the wet bulb assembly and install a new wick for each test.
• b. In order to produce the evaporation necessary for sensor measurement of wet bulb temperature, ensure the air velocity across the wet bulb is not less than 4.6 m/s (900 ft/min).
• c. Because heat from fan motors may affect temperature readings, do not install wet and dry bulb sensors close to the discharge side of any local fan or blower used to create the requirement of paragraph 4.2.1b.

4.2.2 Circulation

Maintain free circulation of air around the test item and keep the contact area of fixtures supporting the test item to a minimum.

4.2.3 Steam

Do not inject steam directly into the test chamber working space where it may have an adverse effect on the test item and microbial activity.

4.2.4 Unless Otherwise Specified

• a. Use only reagents that conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society, where such specifications are available.
• b. Use water as described in Part One, paragraph 5.16. The intent is to not introduce contaminants or acidic/alkaline conditions that may affect the test results.

4.3.1 Interruption Due To Chamber Malfunction

• a. General. See Part One, paragraph 5.11, of this Standard.
• b. Specific to This Method. The fungus test, unlike other environmental tests, involves living organisms. If the test is interrupted, the fact that live organisms are involved must be considered. Observation of the control test strips should be performed weekly in order to determine corrective action.
• (1) If the interruption occurs during the first seven days of the test, restart the test from the beginning with either a new or cleaned test item. If the interruption (such as a short power outage) does not produce drastic drops in humidity (less than 90 percent RH) and temperature (less than 28 °C (82 °F)), continue the test and add at least twelve hours to the final test time.
• (2) If the interruption occurs between 8 and 25 days of testing, examine the control strips for evidence of fungus growth. The control strips should be observed weekly (through the glass windows or doors without opening the chamber), if possible. Labs should make a point of hanging at least one additional control in an easily visible location in the chambers. If the controls exhibit viable growth but there is no evidence of fungus growth on the test item, follow the guidance given below.

MIL-STD-810H

METHOD 508.8

• (3) If the interruption occurs after 25 days of testing, examine the test item for evidence of fungus growth. If the test item is bio-susceptible, there is no need for a retest. If the controls exhibit viable growth but there is no evidence of fungus growth on the test item, follow the guidance given below.
• (a) Lowered Temperature. A lowering of the test chamber temperature generally will retard fungus growth. If the relative humidity has been maintained, reestablish the test conditions and continue the test from the point where the temperature fell below the prescribed tolerances. If not, see paragraph 4.3.1.b(3)(c) below.
• (b) Elevated Temperature. Elevated temperatures may have a drastic effect on fungus growth. A complete re-initiation of the test may be required if one of the following conditions exist:
• (1) The temperature exceeds 40 °C (104 °F).
• (2) The temperature exceeds 32 °C (90 °F) for 4 hours or more. A trained microbiologist can determine if the conditions warrant a restart.
• (3) There is evidence of deterioration of the fungus growth on the control strips.

Otherwise, reestablish test conditions and continue the test from the point of interruption.

• (c) Lowered Humidity. A complete retest may be required if one of the following conditions exist:
• (1) The relative humidity drops below 50 percent.
• (2) The relative humidity drops below 70 percent for 4 hours or more.
• (3) If the relative humidity drops between 70-90 percent for more than 24 hours, restart the test if there is evidence of fungal deterioration on the strips.
• (4) There is any evidence of deterioration of the fungal colonies on the control strips. Consider use of newly prepared control strips after any test interruptions to aid in identifying new/continued growth.

Otherwise, reestablish test conditions and continue the test from the point of interruption.

4.3.2 Interruption Due To Test Item Operation Failure

Failure of the test item(s) to function as required during operational checks during or following testing presents a situation with several possible options.

• a. The preferable option is to replace the test item with a “new” one and restart from Step 1.
• b. A second option is to replace / repair the failed or non-functioning component or assembly with one that functions as intended, and restart the entire test from Step 1.

4.4.1 Cleaning

Although it is preferable to use a new test item, a used item may be substituted. Cleaning (not sterilizing) can consist of wiping with a damp (water or other solution) cloth or following the test item cleaning instructions. If a solution other than water is used during the cleaning process, document the cleaning solution used in the test report. The cleaning shall be conducted at least 72 hours before test initiation. Prepare the test item in accordance with paragraph 4.5.1.

4.4.2 Miscellaneous

• a. This Method is designed to provide optimal climatic conditions and all of the basic inorganic minerals needed for growth of the fungal species used in the test. The group of fungal species was chosen for its ability to attack a wide variety of materials commonly used in the construction of military materiel. Optional species may be added to the inoculum, if required (see paragraph 2.2.2).
• b. This test must be performed by trained personnel at laboratories specially equipped for microbiological work.
• c. The presence of moisture is essential for spore germination and growth. Generally, germination and growth will start when the relative humidity of the ambient air exceeds 70 percent. Development will become

progressively more rapid as the humidity rises above this value, reaching a maximum in the 90 to less than 100 percent relative humidity range.

• d. The specified temperature of 30 ± 2 °C (86 ± 3.6 °F) is most conducive to the growth of the test fungi.
• e. Control items specified in paragraph 4.4.3.3 are designed to:
• (1) Verify the viability of the fungus spores used in the inoculum.
• (2) Establish the suitability of the chamber environment to support fungus growth.
• f. Although this procedure can provide information on the susceptibility of materials to fungus growth, the testing of materials and parts will not reveal potential fungus growth situations in materiel that can result due to the complexities involved in assemblages. Examples are induced conditions created by coatings and protective wrappings, deterioration of protective coatings due to bi-metallic reactions, and other situations that would not be encountered with the testing of components.

4.4.3 Preparation for Test

4.4.3.1 Preparation for Mineral Salts Solution

• a. Using clean apparatus, prepare the mineral salts solution to contain the following:

• b. Ensure the pH of the mineral salts solution is between 6.0 and 6.5.
• c. Sterilize the solution for at least 30 minutes at 121 °C (250 °F) and 15 psi or according to the autoclave manufacturer’s recommendations. To avoid precipitation of the solution during heating, separate the (KH2PO4) and (K2HPO4) from the rest of the solution. After autoclaving, combine the solutions and bring to final volume after cooling. If another sterilization technique is used in lieu of autoclaving (such as filter sterilization), state the procedure used in the test report.

4.4.3.2 Preparation of Mixed Spore Suspension

NOTE - PRECAUTIONS: Although the exact strains of fungus specified for this test are not normally considered to present a serious hazard to humans, certain people may develop allergies or other reactions. Therefore, use standing operating procedures/good laboratory housekeeping techniques for safety. Also, use only personnel trained in microbiological techniques to conduct the tests.

• a. Use aseptic techniques to prepare the spore suspension containing the test fungi determined from paragraph 2.2.2. All hardware used should be sterile (as packaged or autoclaved) and the prep area should be disinfected. A bio-safety cabinet should be used to eliminate cross-contamination of spores or their release to the surrounding laboratory air.
• b. Maintain pure cultures of these fungi separately on an appropriate medium such as potato dextrose agar, but culture Chaetomium globosum on strips of filter paper overlaid on the surface of mineral salts agar. Prepare the mineral salts agar by dissolving 15.0g of agar in a liter of the mineral salts solution described in paragraph 4.4.3.1.

MIL-STD-810H

METHOD 508.8

NOTE: Do not keep the stock cultures for more than 6 months at 6 ± 4 °C (42 ± 7 °F). After that time, prepare subcultures and use them for the new stocks.

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• c. Visually verify the purity of fungus cultures before the test.
• d. Make subcultures from the pure stock cultures and incubate them at 30 ± 2 °C (86 ± 3.6 °F) and greater than 90 but less than 100 percent relative humidity for 10 to 21 days. Most fungi will develop within 10 to 14 days and may show signs of deterioration after longer incubation.
• e. Prepare a spore suspension of each of the required test fungus by pouring into one subculture of each fungus 10 ml of a sterilized aqueous solution containing 0.05g per liter of a nontoxic wetting agent such as sodium dioctyl sulfosuccinate or sodium lauryl sulfate. There are several ways to aseptically harvest the necessary quantity of fungal spores. One way is to gently scrape the surface growth from the culture of the test organisms. Pour the spore charge into an appropriately sized sterile, Erlenmeyer flask containing sterilized water and glass beads (10 to 15 glass beads with a diameter of 4mm to 6mm has proven sufficient).
• f. Shake the flask vigorously to liberate the spores from the fruiting bodies. Filter as needed using sterile glass wool (i.e., Pyrex 3950 filtering fiber) in a sterile glass funnel to remove mycelium, but leave spores in solution. Centrifuge the filtered spore suspension and discard the supernatant liquid. Wash the pure suspensions with sterile water until the supernatant is clear.
• g. Dilute the final washed residue with the sterilized mineral-salts solution in such a manner that the resultant spore suspension contains 1,000,000 ± 20 percent spores per milliliter as determined with a counting chamber.
• h. Repeat this operation for each organism used in the test.
• i. Perform a viability check for each organism in accordance with paragraph 4.4.3.3.
• j. Blend appropriate volumes of the resultant spore suspensions to obtain the final mixed spore suspension.
• k. If a different technique is used to harvest the spores, state the procedure in the test report.

NOTE: Use a freshly prepared spore suspension. If not freshly prepared, it should be held at 6 ± 4 °C (42 ± 7 °F) for not more than 14 days.

4.4.3.3 Control Items

Two types of control tests are required. Using the following procedures, verify the viability of the spore suspension and its preparation, as well as the suitability of the chamber environment.

• a. Viability of spore suspension.
• (1) Before preparing the composite spore suspension, inoculate sterile potato dextrose (or another nutrient agar) plates with 0.2 to 0.3 ml of the spore suspension of each of the individual fungus species. Use separate agar plates for each species.
• (2) Distribute the inoculum over the entire surface of the plate.
• (3) Incubate the inoculated potato dextrose agar plates at 30 ± 2 °C (86 ± 3.6 °F) for 7 to 10 days.
• (4) After the 7 to 10 day incubation period, check the fungus growth.

NOTE: The absence of copious growth of any of the test organisms over the entire surface in each container will invalidate the results of any tests using these spores.

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• b. Verifying test chamber environment.

MIL-STD-810H

METHOD 508.8

• (1) To ensure proper conditions are present in the incubation chamber to promote fungus growth, install control strips into the chamber which have been soaked in the following prepared solution:

Use HCl and NaOH to adjust the final solution pH to 5.3 (this solution is not sterilized but used immediately in preparation of the control strips).

• (2) Prepare control strips from unbleached, plain weave, 100 percent cotton cloth, such as MIL-T-43566. (Type I, Class I only, commercially available). Sterilize the control strips prior to soaking in the above solution. After the strips are thoroughly wetted, remove the excess liquid from the strips and hang them to dry. (Use only strips devoid of fungicides, water repellents and sizing additives. To aid in removing any possible treatment materials, recommend boiling in distilled water (not required if using MIL-T-43566, Type 1, Class I)).
• (3) Place the strips vertically within the chamber close to and bracketing the test items to ensure the test strips and test items experience the same test environment The width of the strips should be such that they can be easily viewed for growth during testing (recommend at least 1.9 cm (0.75 inches width).

4.5.1 Preparation for Incubation

• Step 1 Assure the condition of the items to be tested is similar to their condition as delivered by the manufacturer or customer for use, or as otherwise specified. Accomplish any cleaning of the test item at least 72 hours before the beginning of the fungus test to allow for evaporation of volatile materials.
• Step 2 Install the test item in the chamber or cabinet on suitable fixtures, and remove any covers. (see note and Step 5 below).
• Step 3 Hold the test item in the test chamber at 30 ± 2 °C (86 ± 3.6 °F) and a RH of at least 90 percent but less than 100 percent for at least four hours immediately before inoculation.

Step 4 Inoculate the test item and the cotton fabric chamber control items with the mixed fungus spore suspension by spraying the suspension on the control items and on and into the test item(s) (if not permanently or hermetically sealed) in the form of a fine mist from an atomizer or nebulizer. Ensure personnel with appropriate knowledge of the test item are available to aid in exposing its interior surfaces for inoculation.

NOTE: In spraying the test and control items with composite spore suspension, cover all external and internal surfaces that are exposed during use or maintenance. If the surfaces are non-wetting, spray until drops begin to form on them.

Step 5 In order for air to penetrate, replace the covers of the test items without tightening the fasteners.

Step 6 Start incubation (paragraph 4.5.2) immediately following the inoculation.

MIL-STD-810H

METHOD 508.8

4.5.2 Incubation of the Test Item

Step 1 Except as noted in Step 2 below, incubate the test items at constant temperature and humidity conditions of 30 ± 2 °C (86 ± 3.6 °F) and a relative humidity of at least 90 percent but less than 100 percent for the test duration (28 days, minimum).

Step 2 Inspect the growth on the control cotton strips after 7 days to verify the environmental conditions in the chamber are suitable for growth. Verify that at least 90 percent of the surface area of each test strip located at the level of the test item is covered by fungus (fungus may not have developed color. At 14 days, fungus can be readily seen and 90% coverage of strip should be verified). If it is not, repeat the entire test with the adjustments of the chamber required to produce conditions suitable for growth or check spore viability. Leave the control strips in the chamber for the duration of the test and visually check the growth weekly (observe through window).

Step 3 If the cotton strips show satisfactory fungus growth after 7 days, continue the test for the required period from the time of inoculation as specified in the test plan. If there is no increase in fungus growth on the cotton strips at the second inspection interval (day 14) of the test as compared to the initial 7-day results, the test is invalid and see paragraph 4.3.1for guidance.

Step 4 If the end of designated incubation time falls on a holiday or non-work day due to a scheduling issue or extension of the test due to environmental parameter outliers, extend the test time as needed and note all deviations in the final report.

4.5.3 Inspection

At the end of the incubation period inspect the test item immediately and if possible, within the chamber with the circulation fans off. If the item is removed from the chamber to conduct the inspection, recommend completing the inspection within 4 hours. If the inspection takes longer than 4 hours, return the item to the chamber or a similar humid environment for a minimum of 2 hours prior to completing inspection. Record the results of the inspection.

4.5.4 Operation/Use

(To be conducted only if required.) If operation of the test item is required (e.g., electrical materiel), conduct the operation in the inspection period as specified in paragraph 4.5.3. Ensure personnel with appropriate knowledge of the test item are available to aid in exposing its interior surfaces for inspection and in making operation and use decisions. Disturbance of any fungus growth must be kept to a minimum during the operational checkout. If the test item fails to operate as intended, see paragraph 5, and follow the guidance in paragraph 4.3.2.

WARNING: Because of the potential hazardous nature of this test, operation/use by personnel with appropriate knowledge of the test item will be performed under the guidance of technically-qualified personnel (e.g., microbiologists). Appropriate personal protective equipment (PPE) must be worn.