[PAGE 1]
At Acqunt of Date of Original PerieáTime ExtendadAllotnon Autdorlzstion Covered. To uober Ogligatien 3125.1390.5902 20 629.44 1Dec1962

[PAGE 2]
RAOIA 282 FOMH 4.65

[PAGE 3]
CRRTIFICATION

This ié to certify that I bave received an accomtiag fras MULTRA Sub-project  1h3 for the period 1 Jamuary 1963- 22 October 1965. The accounting reflects expenses in the amcut of \$39,823.29 against the remainiag grant balance of \$39,836.00. The residual balance of \$12.71 should be written ofr as boing iapracticle for collection or dotailed accouting.

The accounting statement and peyment certificatioa vill be retained by TSD and will be made availeble for review in TSD if necessary.

I cortiry that satisfactory servicss representpd by the accounting have been received and that to the best of my kaovladge and belief tho fumda expended vere for the purposes authorized by the project approvel.

1413-3

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Continued on next page.

[PAGE 6]
Ac a c   to ny pPp      . c      c

[PAGE 8]
MEHORANDUN FOR C/TSU/FAES SUBJECT MKULTRA, Subproject No. 143

Under the authority sranted in-the moncrandun dated 13 April l95. from the DCI to thé D0/A. and the'extersion-of this suthority ia subsequeat menoranda, Subproject 143 has beon approyed, and \$20,800.00 of the over-all Project:WUEfRA-funde have been ohligatedo-cover tne subproject'a ezpenses and should be charsed tocostoeter4125-1390-3902

Concur:

131 Asst D&E

Concur:

APPROVED FOR OBLIGETION OF FUNDS:

Date:.

Distribution: Orig & 1 - Addresses 2TSD/

GROVPI

Excluded from automatis dovngrading

[PAGE 11]
"xy ter  so  ans gr con dy d  spe s or Boroy L  /

[PAGE 12]
A Tr       n t    se at r Lr o   p  2 t f96t Lr    g/r d

[PAGE 13]
Bnotified us that you have, requested a detailed statement of his grant frout We have prepared the detailed statement which you require.

[PAGE 14]
L   L Ith you this morning and it was agreed that: You would again contact tho contractor ro the \$18,20 unexpended balance reflected in the accounting for the period ending August 31, 1962, and not carried forward to the next grant year;

.the current grant poriod would be extended in time only to 30 June 1966, giving the grantee the:opportunity to expond in the field the \$123.69 residue, as requested in the Japuary 10th, 1966 Mthe original of this letter from letter is enclosed herewith)

Also enclosed 1s the original of the Pebruary l0th, which forvarded 1966 letter fron tho this granteo'a accounting for exponditures during the period January lat, 1903 through October 22nd, 1965.

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MEHCRANDUM FOR : Chief, TSD/BB

SUBJECT :-Delinquent Accountings MULTRA, Subproject 143

1. Reviev of MKULTRA, Subproject l43 financial records indicate that accountings for grants are now past due. It is requested that your office initiate action to obtain the delinquent accountings.

2. For your information funds have beea advanced to the grantee

as follows.

Authorization 1 \$19,836.00
Grant period 1 Jan.- 31 Dec. i963
Authorization 2 \$20,000.00
Grant period 1 Jan.-31 Dec, 1964

19 January 1966

Distribution: Original& l- Addressee

[PAGE 16]
Receipt is hereby acknowledged of dated 0

[PAGE 17]
DATE RENFWED

During the past year, the investigators have studied a total of 57compounds for their capacity to stimulate bacteria. These 57 compounds-consisted of 34 phenols, 13 amines, 5 aldehydes and ketones, 3 quaternary ammonium compounds and-2 unclassified chemicals,This study has shown that the phenolic chemicals are-the-best compounds for stimulating bacteria. Of this group they-have noted that Q-phenyl-phenol,p-tert-butylphenol and nonyl phenol are the best compounds studied so far.

They have-been-able to produce as much as-a four-fold increase in microbial growth using these compounds.This degree of stimulation is not as great as they had hoped it would be. For this reason, during the past three weeks they changed their line of approach to the problem.

They have produced a product consisting of phenol, casein hydrolyzate,: yeast extract\_and peptone which has-a much greater stimulating capacity.It appears that concentrations as small as 50 ppm will give them a consistant 10 fold:increase-in growth. The deterioration of the cutting fluid is-greater in three days than they-have ever observed in their studies-with these products at any time. In addition, as yet unpublished information from this laboratory indicates that this produce should neutralize practically any known biocide that:might-be added to cutting fluids.

It is believed that they have arrived at:a point where dynamic progress can be anticipated, In view: of the high proudcitivity and promise of results that will be of direct benefit to the operational aspects of DDP. It will be the purpose of the renewed project to combine the two principles of bacterial stimulation (the use of subeffective concentrations-of inhibitors and\_the use of non-specific protective compounds) to determine the ultimate stimulation to be expected in\_ cutting fluids.: More emphasis will be given to actual degradative processes rather than stimulation of growth per-se.-In addition has agreed to investigate microbial fouling of diesel cooling systems in conjunction with!his other work.

PROJECT CRYPTO

MKULTRA #143

PRINCIPAL INVESTIGATOR

:1 January 1964

MKULTRA

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AVCa ra     y . sy y    acs s V S S .350#4

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COVERT ACTION SUPPORT Sabotage

Type of ContractMKULTRA Date Initiated 1 January1964 FY 63 - \$20,629.44 Cost FY\_64\~\$20.800,00 Completion Date. 1 year

Purpose: The objective of the first year was acnieved. An approach to the stimulation of soluble-oil degradation was proved feasible. In addition a eystem for negating microbial inhibitors was developed. These two accomplishments appear to be the first significant breakthrough for microbial degradation of materials. It is planned to study further the limitations of these approaches and their applicatioi to a variety of praotical situations during the coming year.

Status: Porformance has been excellent. Has rogressed beyond expectations. Current in fiscal matters.

Requirement: Generated by undocumented requests from DDP units and divisions as well as necessity for TSD to naintain a quick delivery capability to meet anticipated future operational needs.

Date 8 January 1964

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\$20,000.00. To this sum must be added \$800.00 representing a four per cent service charge to the The will function as-cut-out and cover for this grant.The cover title of the projectis

5. Total cost of the project wi11 not exceed \$20,800.00 Charges should be made againstA1lotment No.4125-1390-3902.

6. The wi11 request the to submit-a summary accounting of monies received and to return any unexpended grant funds at the end of the project year.

7.Title to any permanent equipment purchased by funds granted the shall be retained by them in lieu of higher oyerhead rates.

8. :It was mutually agreed that documentation and accounting for travel expenses which are reimbursable by the hall conform with the accepted practices of

Attachments: Mote:-Due-date for accounting of Proposal & budget previous. year's funds is 31 mwch1964.

Distribution: Original only

UUAKULTRA

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MEMORANDUM FOR : THE RECORD

SUBJECT

1. The purpose of subprojeet 143 is to continue support of the work of at the 3 on an approachto microbial deteriorationof materials.

2. During the first year a system was developed which allows à l0 fold stimulation of the microbial deterioration of soluble oil coàlants (as a type system) using extremely small quantities of active material. In addition a system was found:which apparently serves to inactivate most microbial inhibitors in such a product. This was much more of an accomplishment than was expected. For the" coming year it is planned to explore the limitations of these approaches as they apply to cutting oils and other petroleum based products It is hoped-that these techniques will be applicable acrossthe-board:to-all sabotage or harassrent systems involying microorganisms, promoting a more deiverable package to the operator.

3. principal investigator,\_hasbeen cleared through\_SECRET. 1

8 January 1964

SRMKULTRA

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dage: 8 January 1964

MEHORANDU FOR:C TSD FASS

Under the:authority grautad in the nescranduu datod 18. Aoril 1953 from the ICI to tha DD/A. and the extension of chis-authorliy in qub sequont reaoranda, SUD co 143 bs5 beer apyrved and \$20,800.00 of the over-all project u 2A funds é subproject'e expenses and sbould b9 chsigad 0 coat cen:4125-1390-3902

IC caai: yo0 4125-1320-390

APPROVED FOR.OBLIGATION OF FUNDSZ

GROUP\_I

Excluded TrOn 0u doengvadi 122 3

Date:

Distributica: Orie E 1  àddressee-2 \~ T5, BB

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During the past year we have studied a total of 57 compounds for their capacity to stimulate\_ bacteria. These 57 compounds consisted of 34 phenols, 13 amines, 5 aldehydes and ketones, 3 quaternary ammonium compounds and 2 unclassified chemicals. This study has shown that the phenolic chemicals are the best conpounds for stimulating bacteria. Of this group we have noted that Q-phenyl-phenol, p-test-butylphenol, disec-butylphenol,:dodecyl pehnol, 2,4-dimethyl-6-test-butylphenol and nonyl phenol are: the best compounds studied so far.

We have been able-to produce as much as a four-fold increase in microbial growth using these compounds. This degree of stimulation is not as great as we had hoped it would be. For this reason, during the past three weeks we changed our line of approach to the problem.

We have produced a product\_consisting of phenol, casein, hydrolyzate, yeast extract and peptone which has a much greater stimulating capacity. It appears that concentrations as small as 50 ppm will give us a consistant 10 fold increase in growth. The deterioration of the cutting fluid is greater in three days than we have ever observed in our studies with these products at any time. In addition, as yet unpublished information from this laboratory indicates that this product should neutralize practically any known biocide or stat that might be added to cutting fluids.

We feel that we now bave arrived at a point where we can\_make\_some dynamic progress. During the coming year we hope to:

1. Study additional chemicals and media products for their stimulatory capacity.

2. Determine the best ratios of the media\_components\_and generally improve the formulation.

3. Determine the effect of the formulation on other petroleum products,

4. Determine if the formulation has the capacity to neutralize antimicrobial agents.

5. Begin work on how the formulation stimulates microorganisms.

6. Determine the effect of the formulation plus urine on microbial growth.

I believe these points cover the main objectives for the coming year.
Kindest personal regards.

Sincerely yours,

[PAGE 24]
PSTNY

PROPOSED GRANT COST

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27 January 1964

MEMORANDUM FOR THE RECORD

SUBJECT: MKULTRA Subproject\_143

1. At the request of both Chief and Deputy Chief, TSD wascontacted for the purpose of funding this-MKULTRA Subproject through one of the established funding mechanisms controlled by Due pri marily to the fumiding date, it was not possible to accomplish this at this time.

2. Arrahgements will be made with to project our funding requirements for MKULTRA Subprojects on a semi-annual basis. With this lead time, feels that they may be\_capable of funding our total requirements.

3. will meet with todiscuss:other Subprojects funding mechanisms\_at 1115this date.

Exctude1 aotomalls

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COVERT ACTION SUPPORT -Sabotage-

Contractor has-developed a model-systen-for desired-systom. Will now attempt-to apply it on small scale.Current in fiscal matters.

Requirement:Generated by undocumented-requests from DDP units:anddivisions as well-as,necessity-for TSD to maintain a quick delivery:capability to meet anticipated-future operational

Date 5 September 1963

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143-22 RECEIPT

Receipt is hereby acknowledged of E dated January 7, 1963, "drawn on the rayable to B in the amount of \$19,836.00

Receipt is hereby acknovledged of dated January ?, 1963
payable to
in the amount or Date10 1463

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11 December 1962

SUBJECT :MULTRA, Subproject 143

1. The purposc or subproject 143 is to support the work of

on an approach to the stimulation of petroleum

product deterioration.

7 2. Although the model system for these studies will be petroleum products, this appraoch to stimulation of deterióration nay have wide application to all areas of biological sabotage currently of interest to.TSD.

principal investigator, has been cleared 3. throvgh SECRST.

5. Total cost of the project for a period-of one year will not exceed 20,629.44.Charges should te made against-Allotmen't No.

DRAFT

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3125-1390-3902.

Will request the to submit a summary accounting of monies received and to return any unexpended grant funds at the end of the project year. 7. Title to any permaneat equipment purchased by funds granted the Shall be retained by them in lieu of higher over-

head rates.

8. It was-mutually agreed that documentation and accounting for travel expenses which are reimbursable by the shall conform with the accepted practices of that institution.

9. The requirement for a six months' informal accounting on the part of the principal investigator is waived.

TSD/BiologicalWaich

Attachment: Proposal-& Budget

Distribution: Original only

TSD/Biological Branch

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Biographical Sketch

Entire page

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Publications-and Papers:

5 pages

Symposla and Books.

I page

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and can place the following equipment at the disposal

this project: Balances (Sartorius, etc.) Warburg Ultra-centrifuge Ratheon Sonic Oscillator Mickle Disintegrator

Centrifuges(refrigerated, Sorval, International, Ultra-centrifuge, Spinco, etc.)
Sharples Super Centrifuge
Beckman DU 60 Spectrophotometers
ReckmanR 8eckmanB
Coleman Photofluorometer
Refrigerated Warburg
Incubators
Research pH Meters
Shaking Incubators
Kjeldahl Apparatus
Colorimeters

Pilot Plant Fermentors Chromatography Equipment

Isotope laboratory with complete supporting equipment

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To determine if the growth of. microorganisms in petroleum products can be markedly increased by the addition of substatic concentrations of antimicrobial agents

The literature has been carefully searched b determine what s known concerning the use of antimicroblal agents to stimulate microbial growth. To date, only two papers have been found that relate to thiș-topic. relate to this-tonic.

Imshenetskii and Perova (Institute of Microbiology, Academy of Science U.S:S.R., Mikroblologiya, 23, 53-56, 1954) found that cultures of Saccharomyces cerevisiae trained to grow in the presence of

small concentrations of HgCl2 or phenols have much higher fermentation rates than cultures that have not been trained to grow in the presence of these compounds. As little as 50 ppm of Hgcl2 accelerates the production of beer.  Those workers believe that this effect is due to traces of inhibitor in the environment and not because of some change in the physiology of the cells.

reported that 50 ppm of 2,4,6-trichloro phenol could markedly stimulate the growth of bacteria in emulsion oils. In some instances this compound produced slmost a three fold increase in the number of cells in the environment.

found that 25 to 150 ppm of 2,4-dinitrophenol increases the oxidation of C4, C6.. C8. C10 C12, C13. C14, and G6fatty acids. The increase was as much as l00% and was not associated with an increase in

[PAGE 35]
These results imply that two types uistimu produced by substatic concentrations of inhibitors.First, a metabolic stimulation may be produced where the cells do not increase in number but this metabotic rate is increased. Second, the number of cells in the envtronment may be increased by the compound. It remains to be seen whether there is a stimulation in the individual metabolic activity of each cell in this case. There is an increase in activity, however, this may only be due to the greater number of cells.

B UNPUBLISHEO DATA have found that 1000 ppm of/ 2-nitro-l-butyl acetate produce as 3 fold increase in the production of penicillin by Penicillium chrysogenum. PLAN OF ATTACK It is well known that the rate of oxidation of hydrocarbons may be limited by the rate of dufision of the compound across the permeability barrier.It is also well known that surface active agents increase the permeability of the cytoplasmic membrane. Severalyyears developed the idea that the use of surface active ago compounds may increase the accessibility of hydrocarbon substrates to microbial attack and thus speed.up the oxidation-of these compounds. Due to several factors including a lack of funds, this idea has To-date, we-have-studied-3 phenolic never been tested to any extent. compounds. Two of these-compounds produced-stiulation and one did not.

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the oxidation of two different synthetic coolants-and one petroleum type product.We may also use other antimicrobial agents other than those listed above

EXPERIMENTAL PROCEDURE

CULTURES Pure cultures of microorganisms belonging to the Genus
Pseudomonas will be used in this investigation.The cultures
will be isolated from samples of used cutting ftuids obtained in
the Houston area
MEOIA

Whenever posstble, the organisms will be maintatned in cutting fluids. In some instances conventional media such as nutrient broth meat infusion broth, or an inorganic salts medium may be used in this investigation. INCUBATION TEMPERATURE All experiments will be done at 30℃. The length of the incubation period will vary according to the organism and the particular experiment. CHEMICALS

The best grade of chemical obtainable will be used throughout this investigation.

GLASSWARE

All flasks,\_tubes and other glass-equipment will be actd cleaned orior to each experiment.

HYOROGEN ION CONCENTRATTON

The pH of the environment will vary according to the particular experiment. In general, we will let the Ph of the cutting fluid

[PAGE 37]
The cells used in the experiments will be grown on an appropriate medium. They will be harvested atter a suitable incubation perlod and washed three times in isotonic saline. The cells will be suspended in isotonic saline and uniform quantities of this suspension will be used to inoculate the tubes or flasks STERILITY We will employ suitable procedures for sterilizing the media containing the:petroleum products.

CONTROLS Appropriate controls will be included .in each experiment. Each experiment will be done in duplicate and repeated at least once.

CUTTING FLUIDS Three different cutting fluids will be used in thts investigation
consistingofitwo chemical coolants and one petroleum product.
EXPERIMENTAL TECHNTQUE

Each cutting ftuid will be prepared in a il--25 dilution in tap water. One hundred ml of this coolant wilì be placed in each 250 ml Erlenmeyer flask.The number of flasks used in:each experiment will vary from time:to time.The inhibitor will be added -to the series of flasks in varylng concentrations. These flasks will-be sterilized by autoclaving and then inoculated\_ with the test\_organisms.The flasks will be incubated for a\_ period of\_time (probably. two weeks) in\_ a shaking incubator.

At periodic intervals of about  2 days time the number of cells per ml will be:determined for each flask. At the same time the pH of the flask will be \_determined\_and other biochemical tests (such as dehydrogenase activity) may be conducted to determine the degree-of metabolic activitv in the environment. At the end of the exoeriment

determine the pH of the experiment.

[PAGE 38]
to determine the quantity of free oil in the product (this test will be used on the petroleum coolant only). The quantity of free oil present is an excellent index of the degree of attack on the

emulsifler In addition, Warburg studies will be conducted at the optimim
stimulatory concentration determined in the growth expedent to
establish the effect of the antimicrobial agent on oxygen uptake We will study as many compounds as possible during the year

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COST OF PROJECT

900.00 Expendable Equipment & Supplies n. Publication Cost

Typing report, papers,  and purchasing reprints of publications that may arise from this work \$ 400.00