Mueller-Hin­ton Medium

by the STC team

On June 11th, we were ad­dressed in a tweet by mi­cro­bi­ol­o­gist Joe Har­ri­son (@JoeHarrison1): "This seems like a timely topic for a blog or an ar­ti­cle @ASMicrobiology @STCmicrobeblog. Mueller-Hin­­ton agar is used glob­ally and has stan­dard­ized the study of #AMR. Be­fore to­day, I'd never read the story of Jane Hin­ton @citeblackwomen"

Dr. Jane Hin­ton (1919–2003). Source. Fronti­spiece: Co­lonies of Burk­holde­ria pseudo­ma­llei on Müller-Hin­ton agar af­ter 72 hours incuba­tion. Source

Yes, this re­quest res­onates with us. We are well aware that the di­ver­sity among us mi­cro­bi­ol­o­gists is not as it could and should be − in con­trast to the over­whelm­ing di­ver­sity among the mi­crobes that we are so happy to con­sider (pun in­tended). Some­times young stu­dents de­spair that in mi­cro­bi­ol­ogy, and equally in mo­lecular bio­logy, not only im­por­tant sci­en­tific achieve­ments but also lab­o­ra­tory equip­ment, ex­per­i­men­tal tech­niques, and cul­ture me­dia are named af­ter the re­searchers who in­troduced them. It is about show­ing that it was ac­tu­ally peo­ple who found or in­vented some­thing, a mat­ter of re­spect. Yet, it's usu­ally the fam­ily names that stick, which makes the con­tri­bu­tion of women nigh in­vis­i­ble in a male-dom­i­nated field (also in the per­cep­tion of stu­dents). When­ever pos­si­ble, we give the full names of the re­searchers whose work we fea­ture. Michaelis-Menten ki­net­ics? ...was jointly de­vel­oped by Leonor Michaelis and Maud Menten. Kozak se­quence? The con­cept was devel­oped by Mar­i­lyn Kozack. Etcetera.

So, to­day we cel­e­brate Jane Hinton's con­tri­bu­tion to mi­cro­bi­ol­ogy, her work, to­gether with J. Ho­ward Mueller, in the early 1940s on a re­li­able cul­ture medium for Neis­se­ria, which has proved use­ful also for other hu­man pathogens, to this day. You may like to read more about the life and work of Dr. Jane Hin­ton, the first African-Amer­i­can woman to be­come a Doc­tor of Vet­eri­nary Med­i­cine in the United States (1949). For us, the best way to cel­e­brate an ac­com­plished sci­en­tist is by repro­ducing her work in this blog (lightly an­no­tated for a few to­day un­com­mon terms.)

Pub­lished in Exp Biol Med (May­wood), 48(1), 1941, 330−333. DOI: 10.3181/00379727–48-13311

A Pro­tein-Free Medium for Pri­mary Iso­la­tion of the Gono­coc­cus and Meningo­coc­cus.

J. HOWARD MUELLER AND JANE HINTON

From the De­part­ment of Bac­te­ri­ol­ogy* and Im­munol­ogy, Har­vard Med­ical School, and School of Pub­lic Health, and the Boston Dis­pen­sary, Boston, Mass.
*Aided by a grant from the Com­mon­wealth Fund.
 

      It is de­sired to re­port the de­vel­op­ment of a trans­par­ent, re­pro­ducible medium, con­tain­ing no heat-la­bile ma­te­r­ial such as serum, which is suf­fi­ciently sta­ble to with­stand au­to­clave ster­il­iza­tion and stor­age, and which will sup­port sat­is­fac­tory pri­mary growth of ei­ther meningo­cocci or gono­cocci. The lat­ter were used as the test or­gan­isms dur­ing the ex­per­i­men­tal work, since a con­stant source of pri­mary cul­tures of the meningo­coc­cus is or­di­nar­ily not avail­able. The sim­i­lar­ity be­tween the two or­gan­isms, and the re­put­edly greater dif­fi­culty ex­pected in the cul­ti­va­tion of the gonococ­cus, in­di­cated that a medium suit­able for this or­gan­ism should prove equally sat­is­fac­tory for the meningo­coc­cus.

      As a ba­sis for com­par­i­son, the Difco "Pro­teose No. 3 Choco­late Agar" has been used. This me­di­um has been adopted as a stan­dard in cer­tain lab­o­ra­to­ries for the di­ag­no­sis of gon­or­rhoea¹  and one of us (J.H.) was thor­oughly fa­mil­iar with its use.

      The plan as for­mu­lated in be­gin­ning the ex­per­i­ments was es­sen­tially that fol­lowed with consi­derable suc­cess with other or­gan­isms, namely, to se­lect the most suit­able em­pir­i­cal medium avai­lable and at­tempt to break it down to its es­sen­tial com­po­nent parts. The ex­pe­ri­ence of the se­nior au­thor in 1918 with the Gor­don and Hine² pea meal ex­tract agar for meningo­coc­cus car­rier de­tection had shown that this medium, when care­fully pre­pared, was em­i­nently sat­is­fac­tory. It pro­ved, upon trial, to be equally so for pri­mary cul­ti­va­tion of the gono­coc­cus, and it was there­fore cho­sen as the start­ing point in the in­ves­ti­ga­tion. It con­sists es­sen­tially of a tryp­tic di­gest of meat, agar, and a 5% NaCl ex­tract of pea flour, the lat­ter pre­sum­ably sup­ply­ing some es­sen­tial pro­tein ma­te­r­ial.

      The frac­tion­a­tion of the pea ex­tract proved sur­pris­ingly sim­ple, since it could be ac­com­plished by me­chan­i­cal means. The ac­tive por­tion proved to be, not pro­tein, but starch. This might per­haps have been an­tic­i­pated on the ba­sis of Vedder's early work³. Sub­sti­tu­tion of or­di­nary laun­dry starch com­pletely re­placed the growth pro­mot­ing ef­fect of the pea ex­tract. The ef­fect is ev­i­dently due to the starch it­self, rather than to some ad­her­ent im­pu­rity of small mol­e­c­u­lar size. This has been shown in two ways. First, it was found that a va­ri­ety oi starches,-corn, potato, wheat, rice, ar­rowroot, etc. were weight for weight iden­ti­cal in their ef­fect. Sec­ond, rel­a­tively brief hy­drol­y­sis of the starch with ptyalin (α‑amylase) com­pletely de­stroys the growth-pro­mot­ing ef­fect. This oc­curs even be­fore the dis­ap­pear­ance of the blue starch-io­dine test, and in­di­cates that so-called "sol­u­ble starches" are likely to prove un­sat­is­fac­tory or ir­reg­u­lar in their ac­tion. The ex­pla­na­tion for the ef­fect of the starch is not yet clear. Pos­si­bly, as McLeod⁴ has sug­gested in con­nec­tion with the ac­tion of as­citic fluid or serum, it acts as a "pro­tec­tive col­loid" against the in­hibitory ef­fects of amino acids.

      The tryp­tic di­gest of meat was next ex­am­ined. This can be re­placed by a rapidly pre­pared meat in­fu­sion to­gether with a com­plete acid hy­drolysate of ca­sein pre­pared as de­scribed else­where by Mueller and John­son⁵ and Mueller and Miller⁶. This hy­drolysate is now ob­tain­able from the Difco Lab­o­ra­to­ries, Inc., De­troit, Michi­gan, un­der their trade name of "Casamino Acids, Tech­ni­cal." Fur­ther ex­am­i­na­tion of the es­sen­tial com­po­nents of the meat in­fu­sion is now un­der way. Since it is quite pos­si­ble that this will prove to be a la­bo­ri­ous mat­ter, this re­port is be­ing made with the hope that the medium in its present form may prove use­ful to oth­ers.

      It has been tested in par­al­lel with the Difco choco­late agar in a con­sid­er­able se­ries of pri­mary cul­ti­va­tions of the gono­coc­cus at the Boston Dis­pen­sary. The re­sults have been highly sat­is­fac­tory. Colonies are fre­quently larger on the starch medium than on the choco­late. They are eas­ily recog­nizable, par­tic­u­larly with the aid of the ox­i­dase reagent. There is no ap­pre­cia­bly greater ten­dency for over­growth of the plates by other or­gan­isms, and no sig­nif­i­cant dis­crep­an­cies be­tween posi­tives and neg­a­tives have been noted. We have re­cently been for­tu­nate in be­ing able to test the medium on a se­ries of pri­mary iso­la­tions of the meningo­coc­cus from both spinal fluid and naso-pha­ryn­geal swabs of cases and car­ri­ers in Hal­i­fax, Nova Sco­tia. This was made pos­si­ble through the cour­te­sies of Dr. A. R. Mor­ton, City Health Com­mis­sioner and Dr. D. J. MacKen­zie, Di­rec­tor of the Frovin­cial Pub­lic Health Lab­o­ra­tory. Con­sis­tently good re­sults were ob­tained in con­nec­tion with this ma­te­r­ial, and a con­sid­er­able num­ber of cul­tures of a Type I meningo­coc­cus were read­ily se­cured. A cor­rect ap­praisal of the value of this medium can re­sult only from a more rig­or­ous se­ries of tests in the hands of other work­ers as well as our own. The de­tails of the prepa­ra­tion fol­low.

For 1,000 cc Starch Agar.

Add 17 g dry shred­ded agar to 500 cc (= ml) tap wa­ter in a 2 liter flask. Au­to­clave 15 min­utes at 15 pounds to dis­solve.

While still hot, add the fol­low­ing so­lu­tion which may be pre­pared while the agar is be­ing au­to­claved:

Meat in­fu­sion^* 300 cc, Ca­sein hy­drolysate^† 17.5 g, Starch paste^‡ 100 cc, Wa­ter 100 cc. Ad­just pH to 7.4−7.6.

* Meat Infusion—1 pound of meat (chopped lean beef or beef heart), 500 cc wa­ter. Sus­pend meat in wa­ter, bring to ac­tive boil­ing, strain through cheese-cloth and fil­ter thnough pa­per. For rou­tine use, this in­fu­sion may be pre­served in the cold room in stop­pered bot­tles con­tain­ing a few cc of chlo­ro­form.
† Ca­sein Hydrolysate—Quantity spec­i­fied for Difco prepa­ra­tion, Lot 8–64123 of "Casamino Acids, Tech­ni­cal."
‡ Starch Paste—Suspend 1.5 g or­di­nary starch (corn starch or laun­dry starch, not "sol­u­ble starch") in 10 cc cold wa­ter. Pour slowly into 90 cc boil­ing wa­ter, while stir­ring.
 

      Mix and dis­trib­ute at once ei­ther into test tubes (about 20 cc each for pours, 5 cc for slants,) or flasks of 120−200 cc. Au­to­clave not more than 10 min­utes at 10 pounds. Over-au­to­clav­ing spoils the medium. The flasks can be used to pour plates at once. The tubes may be melted in boil­ing wa­ter and used as needed. The medium seems to give best re­sults when not too moist, ei­ther with gono­coc­cus or meningo­coc­cus. For ei­ther or­gan­ism a can­dle jar (im­pro­vised anaer­o­bic cham­ber) must be used, and the cul­tures in­cu­bated at about 36°C for 24 to 48 hours in the case of go­nococcus, or 16 to 24 hours for meningo­coc­cus.
 

Ref­er­ences
¹ Cox, D. F., Mc­Der­mott, M., and Hin­ton, W. A., The Gono­coc­cus and Gono­coc­cal In­fec­tions. Sci­ence Press, 1939, p. 79−82.
² Gor­don, M. H., and Hine, T. G. M., Brit. Med. J., 1916, 2, 678.
³ Ved­der, E. B., J. In­fect. Dis., 1915, 16, 385.
⁴ McLeod, J. W., Wheat­ley, M. B., and Ph­elon, H. V., Brit. J. Exp. Path., 1927, 8, 25.
⁵ Mueller, J. H., and John­son, E. R., J. Im­munol., 1941, 40, 33.
⁶ Mueller, J. H., and Miller, P. A., J. Im­munol., 1941, 40, 21.