THE MICROBACTERIA

I. MORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERISTICS

RAYMOND N. DOETSCH AND MICHAEL J. PELCZAR, JR.

Department of Bacteriology, University of Maryland, College Park, Maryland

Received for publication March 16, 1948

The genus Microbacterium was proposed by Orla-Jensen in 1919. Fourspecies were described, viz., Microbacterium lacticum, Microbacterium flavum,Microbacterium liquefaciens, and Microbacterium mesentericum. The organismsof this genus presented a physiological gradient with the saccharolytic acidformer (M. lacticum) at one extreme and the proteolytic gelatin liquefier (M.liquefaciens) at the other extreme. Later (1921), with reference to the taxonomicposition of this genus, Orla-Jensen stated: "Microbacterium is to be understood asmerely a provisional collective name for gram-positive rods of size a little smallerthan ordinary bacteria. In biological respects some of these rods (Bacillusacidophilus) are closely related to the true lactic acid bacteria, whereas othersapproach the Tetracoccil or the aerobic bacilli." No further detailed investiga-tions were made on this genus for 14 years.

Robertson (1927) encountered M. lacticum in his study of thermoduricorgan-isms isolated from heated milk. His data agreed well with those given by Orla-Jensen (1919) for M. lacticum. An interesting conclusion of Robertson was thestatement that M. lacticum and Lactobacillus thermophilus were identical. How-ever, the description of L. thermophilus by Ayers and Johnson (1924) leaves littledoubt that these organisms are distinctly different.A valuable contribution to our knowledge of the genus was made by Wittern

(1933). Her work confirmed and extended that of Orla-Jensen (1919). A majorportion of her investigation dealt with the taxonomic position of M. mesentericum,which she allocated to the genus Mycobacterium. Jensen (1932) made a mor-phological studv of the genus. His observations led him to the conclusion thatM. mesentericum was a proactinomyces and hence should be Proactinomycesmesentericus. Other recommendations by Jensen were that M. lacticum andM. liquefaciens be designated as Corynebacterium lacticum and Corynebacteriumliquefaciens. Further studies (1934) led him to the conclusion that M. flavumwas actually a Mycobacterium and therefore should be designated as Myco-bacterium flavum.Hansen (1938) found that hydrocyanic acid or iodoacetate inhibited the

respiration of M. lacticum and indicated that the microbacteria differed from thetrue lactic acid bacteria not only in catalase content but also in their hemin con-tent. Speck (1943) took issue with the conclusions of Jensen (1932, 1934),stating that to place the Microbacterium in the genera Mycobacterium or Coryne-

1 Tetracoccus as used by Orla-Jensen refers to acid-producing forms of Micrococcus andSarcina. Much of the acid they produce is acetic acid, and many liquefy gelatin. Thecolonies are generally yellow, orange, or pink.

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bacterium on a morphological basis was not justified. He concluded that anysystem of classification should indicate a close relationship between the generaMicrobacterium, Propionibacterium, and Lactobacillus.

Orla-Jensen (1943), in an attempt to show the taxonomic relationships of themicrobacteria to other groups, was prompted to diagram their very close relation-ship to the corynebacteria and actinomycetes. The Danish master, however,was well aware of the implications involved when he concluded, "Der Voll-standigkeit halber habe ich-mehr oder weniger berechtigeweise-in unseremSystem auch die Mikrobakterien und die Propionibakterien angebracht. Ob esrichtig ist, Corynebakterien und Actinomyceten von den Mikrobakterien und vonBm. bifidum entspringen zu lassen muss die Zukunft lehren."The fifth edition of Bergey's Manual (1939) places Microbacterium in the

second genus of the family Bacteriaceae. The four species recognized by Orla-Jensen (1919) are included in the genus. In the sixth edition of this manual(1948), the genus has been transferred to the family Lactobacteriaceae and is thesecond genus of the tribe Lactobacilleae. M. liquefaciens has been placed in anappendix to this genus and M. mesentericum is classified as Nocardia mesenterica.

It is apparent that conflicting opinions exist both as to the characterizationand the systematic position of these organisms. This investigation presentsdata that may be of some significance in evaluating their taxonomic position.

EXPERIMENTAL METHODS AND RESULTS

Cultures. For the purposes of this investigation, known cultures were receivedfrom the sources indicated in table 1. In addition, original isolations were madeusing the following procedure: Five-ml samples of raw or pasteurized milk werepipetted into sterile test tubes and heated at 61 C for 30 minutes or 73 C for 16seconds. The samples were cooled immediately in an ice bath preceding thepreparation of decimal dilutions. Originally several media were employed forthe isolation of the microbacteria, but, during the course of the investigation, itwas found that a medium of the following composition was at least equivalentand sometimes superior to the others:

Peptone.......... 0.5 gMeat extract............. 0.3 gFructose.......... 0.1gK2HP04......... 0.4gKH2P04 ......... 0.1gDistilled water.......... 100.0 ml

Adjust to pH 7.0 before sterilization

The inoculated plates were incubated at 30 C for 3 days. Microscopicpreparations were made from colonies measuring a few mm or less in diameter.If short gram-positive, nonsporing rods were present, the colony was transferredto litmus milk and incubated at 30 C for 3 days, after which time a subculturewas made to an agar slant. After incubation the agar slant culture was checkedto ascertain purity and then placed under refrigeration for future study. Forty

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cultures were isolated in this manner. Of these, five representatives were in-cluded in this study.

Unfortunately, cultures of M. liquefaciens were not obtained either by theisolation technique described above or from the sources listed in table 1.

Cultural characteristics. Observations on the colonial morphology of themicrobacteria used in this study were made on streak plate cultures grown onDifco tryptone glucose extract agar with skim milk added and incubated 3 daysat 30 C.

TABLE 1Source and designation of cultures

CULTURE DESIGNATION CODE NUMBER SOURCE OF CULTURES

M. lacticum 3 Prof. S. Orla-Jensen,cc tiC 6 Biochemical-Technical Institute,M. flavum 8 Copenhagen, Denmark"s " 9

" lacticum 8180 American Type Culture Collection,it s<8181 Georgetown University,

Washington, D. C.

" lacticum 513 Dr. Johanna Westerdijk,516 Centraalbureau voor Schimmelcultures,

flavum 531 Baarn, Netherlands534

lacticum S-2 Dr. M. L. Speck,30-3 National Dairy Research Laboratories, Inc.,

flavum 342-Si Baltimore, Maryland

lacticum 1PM3 Original isolations,i t 3RM2 University of Maryland,itss<3PMb-9 College Park, Maryland

Alicrobacterium sp. 3RM5it 3RMb-1

The surface colonies of M. lacticum were 0.25 to 0.50 mm in diameter. Theywere punctiform, glistening, smooth, convex, pearl white or gray, translucent,and finely amorphous. They may be described as "dewdrop" colonies. CulturesOJ3, OJ6, S-2, 30-3, 513, 516, 8180, 1PM3, 3RM2, and 3PMb-9 were of this type(figure 1A). Cultures 342-Si and 3RM5 were also of this type but were creamor slightly yellow in color.The surface colonies of M. flavum averaged 2.0 mm in diameter. They were

glistening, smooth, convex, and cream or canary yellow. They were alsogenerally round and possessed an even edge. Cultures 531, 534, OJ8, and OJ9were of this type. The pigment varied from the color of rich cream (OJ8) toa very bright canary yellow (534) (figure 1B).

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>__i_ K[B - E __ iXEk i;. ._.X .s [ __ ____ _K ._ r .s___ t_ :Figure >. A, surface colonies of M. Iacticum S-2 (X5i). B, surface colonies of M.

J£avum OJ8 (X5a). C, D, E, subsurface colonies of M. Iacticum 3RM2 (X57).

The chief difference between the colonies of M. Iacticum and M. flavum ap-peared to be one of color. Generally the colony diameter of M. Iacticumaveraged

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0.5 mm, whereas that of M. flavum averaged 2.0 mm. This was not considereda distinguishing characteristic. Subsurface colonies were usually lens-shaped,although round, "lumpy," and diffuse colonies were occasionally encountered.All of these subsurface colony types could occur from the same culture (figuresIC, D, E). Growth under anaerobic conditions was much slower, requiring 5 to7 days' incubation at 30 C for colony formation, and pigmentation was minimal.

Figure 2. Morphological types of Microbacterium. A, type 1 (M. lacticum 3RM2). B,type 2 (Microbacterium sp. 3RM5). C, type 3 (M. flavum OJ8). D, type 4 (M. lacticum513).

Yeast extract proteose peptone glucose broth cultures of the organismsgenerally showed varying degrees of turbidity from faint to heavy. Most of thecultures formed a stringy, viscid, sediment after 4 days at 30 C. Agar slantcultures showed good surface growth after 2 days at 30 C. In the case of M.lacticum, the growth was greenish white, white, or gray. Many cultures showeda translucent film of growth over the surface of the slant. M. flavum gaverather heavy growth, which was usually cream or canary yellow.

Morphological characteristics. The morphology of the cultures used in this

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RAYMOND N. DOETSCH AND AiICHAEL J. PELCZAR, JR.

study agreed with the descriptions of previous investigators. All of the cultureswere found to be gram-positive (Hucker's modification), nonmotile (hangingdrop), and nonsporeforming. They showed bipolar granulation when stainedwith Loeffler's methylene blue and were non-acid-fast when stained with Ziehl-Neelson's carbol fuchsin and decolorized with acid alcohol. On the basis of cellmorphology, the cultures have been arbitrarily divided into four types as follows:

Figure 3. MIorphological type 1 of microbacteria as show-n by the electron microscope(X ca. 26,000). M. lacticutm strain 3RM12 shadowed with chromium.

Type 1. Short, thin rods (0.5 to 0.7 by 1.4 to 2.1 ,u), occasionally club-shaped,were regarded as the classical type morphology of J11. lacticum. Angular andpalisade arrangements were frequently noted in preparations made from culturesgrown on solid media. Granulation was observed when the cells were stainedwith Loeffler's methylene blue and was pronounced in cultures grown in milk andstained with Newman-Lampert stain. Representatives of this type are shown infigures 2A and 3. The electron micrograph (figure 3) reveals that the cell

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contents are unusually dense. Cultures 3RM2, 1PM3, S-2, 30-3, 3PMb-9, 8180,and 8181 are of this type. Organisms that are probably representative of thistype have been noted by Orla-Jensen (1919), Robertson (1927), Wittern (1933),Dull (1933), and Speck (1943).

T'ype 2. In this type were coccobacillary and wedge-shaped rods as describedby Orla-Jensen (1919) and Jensen (1934). The organisms resembled micrococci.

Figurae 4. Mlorphological type 3 of microbacteria as shown by the electron microg:,raph(X ca. 26,000). M1. flavulm strain OJ8 shadowed with chromium.

In most instances the major axis of the cell was only slightly longer than theminor axis. They had diameters ranging from 0.7 to 1.4 u. Granulation wasapparent in preparations made from milk cultures. Cultures 3RMb-1, 516, 534,342-Si, 3RA15, and OJ6 were of this type (figure 2B).

Type 3. 31. flavutm was representative of this type. The cells measured0.7 to 0.9 by 1.4 to 2.6 A, and none were observed that were 10 , long, a character-istic attributed to them by Orla-Jensen (1919) and Wittern (1933). Cultures

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RAYMOND N. DOETSCH AND MICHAEL J. PELCZAR, JR.

OJ8, OJ9, and 531 were representatives of this type (figures 2C, 4). Here-;againthe electron micrograph reveals the extreme density of the protoplasm.

Type 4. This type had long, thin, filamentous cells. This type has beendescribed by Wittern (1933) as representative of M. mesentericum. Culture 513(labeled M. lacticum) was of this type. It had an unusually long, threadlike

Figure 5. Morphological type 4 of microbacteria as shown by the electron microscope(X 26,000). M. lacticum strain 513 shadowed with chromium.

appearance (30 ,u long) and differed markedly from all cultures used in this study(figures 2D, 5).When microbacteria were grown under anaerobic conditions, their morphology

was not significantly altered. The rod-shaped appearance of some cultures wasmore difficult to detect and, in general, all were more coccuslike in appearance.

Physiological characteristics. Forty physiological reactions were determinedfor each of the cultures, the results of which are presented in table 2. The mediaand methods used conformed as closely as possible to those described in leaflet

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V of the Manual of Methods for Pure Culture Study of Bacteria (1944). Deter-minations of carbohydrate, alcohol, and glucoside fermentations were performedusing a cystine agar base2 to which was added 1 per cent of the test substratepreviously sterilized by filtration. Cultures were incubated at 30 C, and theresults were read at the end of 4 and 8 days' incubation unless otherwise indi-cated. Acid production in skcim milk was determined after 7 days' incubation at

TABLE 2Physiological reactions of the genu-s Microbacterium

MONO- TEl1-SAC- D SAC- POLYSAC- ALCO- ADTOA I TSSCA DSACHA RIDE CHAR- CHARMDS HOLS* ADTOA ICEIA ETIDES* WDES

CULTURECS

41 ~ ~ 4

U.lacticum 0J3 0 OAA A .0 0 000 .000.4PJ60cAAAis00A0 0 01- 0+53A 0051600A00A OAA 0~~~DADAcOOO+0+.O 00. sA

M.uwt 8180 0OoAAA A 0 0 0 0 0 A 0 A 0 0 A 0 O+0+0+ 5.78 0 00.28K A49 id 30-3 0 oAAA A 0 o o o 0 A 0 A 0 0 A 0 0+0+ 0 5.60 0 0 0.19 A9 is 5160oA0AAA A 0 A A A 0 AO0 A 0 0 AOA0+000+5.50 0 00.29K sA

"9 is 81P 0 0 AAA A 0 A 0 0 0 AA0 A 0 0 A0OA 00+ 0 6.10 0 00.31K A

" 3RM200oAAA AOA A AO0AA AOOAO A OA 0+ .00 0 00.37K A" 3PMb-9 0 oAAA A 0 A A A 0 A 0 A 0 0 AAA+0 0 0 5.58 0 00.23K A

513AAAAO0 A 0 A A 0 0 AA A 0 0 0 0 0+00O+5.15 0 00.09 AR" ~~~' 818100oA00 0 o0 0 0 0 o 0 0 0 0 0 00 0+0 0 05.57 0 00.04 0

M.flavum OJ8 0 0AOO 00 0 0 00 0 00 00A 0 000 0 06.85 0+0 R'9 "1 0J9 0 000 0 0 0 O A 00 0 00 00A 0 000 0 06.70 0+0.07 0'9 is 53100oA00 0 0 0 0 0 0 0 0 0 0 0 AOOoooo006.65 0 +0.08 09649 534O0AAAA A A A AO0A AO0A AOAOOOOOO7.00 0 00 0

is 6 M4-SIAAAAA A A A A AOAA 0AOAAA+0 0+5.80 0 00.17 0MicrobacteriumBP. 3RMI5AAAAAOA A AOA AlOO0AOAAAOOOO1 07.08 0 +0.09 Ais 8RMb6-1AAAAA01A A AOA AOO01AlO1AlOJAIOOO 7.00 0 00.14 A

0 - No change or no reaction.A - Acid.+ - Positive reaction or growth.K - Curdled.R - Reduction.* All the cultures studied had the following chsracteristics: glucose, fructose, and mannose were fermented; rham-

nose, inulin, adonitol, and sorbitol were not fermented; catalase was produced; gelatin was not liquefied; and indoleand hydrogen sulfide were not produced.

30 C. Twenty ml of skim milk culture were titrated with 0.1 N sodium hydroxideto the phenolphthalein end point and calculated as lactic acid. The heatresistance of the organisms was determined by subjecting a freshly inoculated vialcontaining 5 ml of buffered glucose yeast extract broth (pH 6.6) to a temperature

2 Cystine 0.5 g, trypticase 20.0 g, agar 3.5 g, sodium chloride 5.0 g, sodium sulfite 0.5 g,phenol red 0.017 g, H20 1,000 ml, pH 7.3. Provided by the Baltimore Biological Labora-tory, Inc., in dehydrated form.

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of 85 C for 21 minutes. Immediately after this treatment, the vials were cooledin an ice bath and subsequently incubated at 30 C for 7 days to determine via-bility.The organisms in the genus Microbacterium employed in this study had the

following physiological characteristics in common: glucose, galactose, fructose,and mannose were fermented; catalase was produced; rhamnose, inulin, adonitol,and sorbitol were not fermented; gelatin was not liquefied and indole and hydro-gen sulfide were not produced.

Because of the fermentation of arabinose, xylose, raffinose, and glycerolit was evident that some of the organisms differed from the typical reactionsof either M. lacticum or M. flavum by fermenting all of these substrates. Thisgroup, which attacked the widest range of carbohydrates, has been tentativelydesignated as Microbacterium sp. Table 3 presents a grouping of the cultures onthe basis of arabinose, xylose, raffinose, and glycerol fermentation. Two cul-tures received from other sources designated as M. flavum (cultures 534 and 342-

TABLE 3Differential biochemical reactions of M. lacticum, M. flavum, and Microbacterium sp.

SPECIES OPF MICKOBACTERIUX ARBINOSE XYLOSE RAPPINOSE GLYCE=OL

M. lacticum (9 cultures) . 0 0 0 0M. flavum (3 cultures) ............... 0 0 0 0Microbacterium sp.B34............................ 0 A A A342-Si ............................ A A A A3RM5........................... A A A A3RMb-1.A A A A

0 = No change.A = Acid.

Si), as well as two original isolations, have been placed with the group designatedas Microbacterium sp. Significantly, all four of these cultures are of morpho-logical type 2. The group designated as Microbacterium sp. attacks the widestrange of carbohydrates with the production of acid. M. lacticum is compara-tively less active in this respect, whereas M. flavum attacks the least number ofcarbohydrates. None of the microbacteria produced gas from carbohydratesdetectable by the usual cultural techniques.The microbacteria are weakly proteolytic, if at all, since they neither liquefied

gelatin nor digested the casein of unneutralized skim milk. Hydrogen sulfideand indole were not produced. Reduction of nitrate by M. lacticum was variable,but none of the typical M. flavum strains were able to effect this change. It isapparent that this characteristic is of limited value in identifying species ofMicrobacterium.Although M. lacticum was reported to be able to survive 85 C for 2' minutes

in milk, only 3 of the cultures used in this study were able to survive this treat-ment in yeast extract proteose peptone broth (pH 6.6). Undoubtedly, the milk

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itself enhances the thermal resistance of the organisms. Although valuable forisolation purposes, thermal resistance does not appear to be a satisfactorycharacteristic for classification of the microbacteria. Determinations of thistype present many difficulties that have been emphasized by Rahn (1945).

Although Orla-Jensen (1919) reported that M. flavum grew as a flaky precipi-tate in 10 per cent salt broth, this characteristic was not observed. Growth inthe salt broth appeared as a viscous sediment similar to that which appeared inthe salt-free broth.

SUMMARY AND KEY

From the foregoing data the following characterization of the microbacteria ispresented:

(1) Microbacterium sp. Gram-positive, nonsporing, nonmotile, wedge-shapedor coccobacillary rods (0.7 to 1.4 ,u diameter). Non-acid-fast.Form small surface colonies about 1 mm in diameter. Colonies smooth,

round, convex, glistening, white or gray. Internal structure amorphous.Catalase-positive.Acid in litmus milk, no coagulation.Gelatin not liquefied.Indole not formed.Hydrogen sulfide not formed.Acid from arabinose, xylose, glucose, galactose, fructose, mannose, cellobiose,

lactose, melibiose, sucrose, trehalose, raffinose, and glycerol.Aerobic, facultatively anaerobic.Optimum temperature 30 C.(2) Microbacterium lacticum. Gram-positive, nonsporing, nonmotile, short

rods (0.5 to 0.7 by 1.4 to 2.1 ,h). Non-acid-fast. Granulated when stained withmethylene blue. Characteristic angular and side-by-side arrangements inpreparations made from solid media. At times appear coccobacillary or wedge-shaped.

Colonies same as Microbacterium sp.CataJase-positive.Acid in litmus milk; coagulation variable.Gelatin not liquefied.Indole not formed.Hydrogen sulfide not formed.Acid from glucose, galactose, fructose, mannose, maltose, dextrin, and usually

starch. Fail to ferment arabinose, rhamnose, xylose, melibiose, raffinose, andglycerol.

Aerobic, facultatively anaerobic.Optimum temperature 30 C.(3) Microbacterium flavum. Gram-positive, nonsporing, nonmotile, short

rods (0.7 to 0.9 by 1.4 to 2.6 1A). Non-acid-fast. Granulated when stained withmethylene blue. May appear coccobacillary or wedge-shaped.

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Form small surface colonies about 2.5 mm in diameter. Colonies smooth,round, convex, glistening, cream or canary yellow.

Catalase-positive.Litmus milk unchanged.Gelatin not liquefied.Indole not formed.Hydrogen sulfide not formed.Acid from glucose, fructose, mannose, and mannitol. No acid from arabinose,

rhamnose, xylose, raffinose, dextrin, starch, or glycerol.Aerobic, facultatively anaerobic.Optimum temperature 30 C.The following key has been constructed that may aid in the identification of

the microbacteria.I. Acid from glycerol and raffinose

A. Acid from arabinose and xylose1. Microbacterium sp.

II. No acid from glycerol and raffinose.A. Acid from maltose and starch

2. Microbacterium lacticumB. No acid from maltose and starch

3. MicrobacteriumflavumACKNOWLEDGMENTS

The authors wish to express their appreciation to Dr. R. W. G. Wyckofffor the electron micrographs presented in this paper and to Mr. G. W. Eastmentand Mr. H. A. Newsom for the remaining photographs depicting the colonial andmorphological characteristics of the microbacteria.

REFERENCES

AYERS, S. HENRY, AND JOHNSON, WILLAM T. 1924 Studies on pasteurization. XII.Cause and significance of pin-point colonies from pasteurized milk. J. Bact., 9, 285-300.

BERGEY, D. H., et al. 1939 Manual of determinative bacteriology. 5th ed. Williams &Wilkins, Baltimore, Maryland.

BREED, R. S., et al. 1948 Bergey's manual of determinative bacteriology. 6th ed. Wil-liams & Wilkins, Baltimore, Maryland.

Com. Bact. Tech. Soc. Am. Bact. 1944 Manual of methods for pure culture study ofbacteria. Leaflet V. Biotech Publications, Geneva, N. Y.

DtILL, ANTON 1933 Diastase bildene Bakterien (unter besonderer Berticksichtigung derMilchsaurebakterien) und Hefen. Zentr. Bakt. Parasitenk., II, 88, 81-124.

HANSEN, P. ARNE 1938 The respiration of the rod-shaped lactic acid bacteria. Zentr.Bakt. Parasitenk, II, 98, 289-298.

JENSEN, H. L. 1932 Contributions to our knowledge of the Actinomycetales. IV. Theidentity of certain species of Mycobacterium and Proactinomyces. Proc. Linnean Soc.N. S. Wales, 57, 364-376.

J15NSEN, H. L. 1934 Studies on saprophytic mycobacteria and corynebacteria. Proc.Linnean Soc. N. S. Wales, 59, 19-61.

ORLA-JENSEN, S. 1919 The lactic acid bacteria. Copenhagen, Denmark.

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ORLA-JENSEN, S. 1921 The main lines of the natural bacterial system. J. Bact., 6, 263-273.

ORLA-JENSEN, S. 1943 Die echten Milchsaurebakterien. Munksgaard, Copenhagen.RAHN, OTTO 1945 Physical methods of sterilization of microorganisms. Bact. Revs., 9,

1-47.ROBERTSON, A. H. 1927 Thermophilic and thermoduric microorganisms with special

reference to species isolated from milk. III. Description of the non-spore-forming,thermoduric organisms isolated. N. Y. Agr. Expt. Sta., Tech. Bull. 131.

SPECK, M. L. 1943 The genus Microbacterium. J. Dairy Sci., 26, 533-543.WIrrERN, ANNA 1933 Beitrage zur Kenntnis der "Mikrobakterien" Orla-Jensen. Zentr.

Bakt. Parasitenk., II, 87, 412-446.

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