Durham Tubes Research Articles

Evaluation of Methods for Determination of Spore-Formers Responsible for the Late Gas-Blowing Defect in Cheese

Late gas-blowing is a defect of European specialty cheeses caused by certain spore-forming bacteria of the genus Clostridium. Five most probable number methods for detecting spores were evaluated: a modified Weinzirl, NIZO-Ede, Fryer/Halligan, BB-lactate, and the BB-lactate with modifications using pure cultures of C. butyricum, C. sporogenes, and C. tyrobutyricum inoculated into sterile homogenized milk. Tubes containing media of the different methods were incubated for 14 d at 32°C for C. butyricum and 37°C for C. sporogenes and C. tyrobutyricum. The modified Weinzirl and NIZO-Ede methods took the longest time to indicate gas formation (blowing) and detected the spore-formers less frequently. The Fryer/Halligan and BB-lactate methods, which contain lactate, detected spores quicker. No apparent differences were observed between the Fryer/Halligan and BB-lactate methods. The BB-lactate method was modified by making the medium semisolid by addition of .2% agar, eliminating the use of Durham tubes, and using an agar plug rather than paraffin. These modifications resulted in a simpler, more sensitive method.

Selection of thermotolerant yeast strains for simultaneous saccharification and fermentation of cellulose

A total of 58 yeast strains from 12 genera were assayed for their ability to grow and ferment carbohydrates in standard Durham tube test at 40, 43, and 46 degrees C. Based on the kinetic parameters for glucose fermentation in shaken flask cultures, the strain Fabospora fragilis CCY51-1-1 was chosen for further studies. It reached about 56.0 and 35.0 g ethanol/L from approximately 140 g glucose/L at 43 and 46 degrees C in less than 48 h, respectively. Trichoderma reesei cellulase preparation (400 FPU/L) had not distinct effect on the ethanol yield and biomass production by the selected strain in the first 12 h fermentation at 46 degrees C. Later a negligible decrease in both yields was observed. It was found that Fabospora fragilis did not grow or produce ethanol at 46 degrees C as tho initial ethanol concentration overcame 40 g/L.

A routine method and test paper method for the differentiation between frozen-thawed and unfrozen fish.

We developed a simple routine method and test paper method for differentiating frozen-thawed fish from unfrozen one. The suggested procedure consists of two steps; (i) incubation of a drop of fish blood with a fluorescent substrate in a Durham tube for 10 min at room temperature, (ii) ter-mination of the reaction by adding an alkaline buffer and observing on the color change of the blood in Durham tube under a UV-lamp. For unfrozen fish, the blood shows red while it shows blue for frozen-thawed fish. The method requires only 10μl of the blood and about 15 min for the entire procedure. Based on the routine method, a test paper method was developed. The differentiation can be performed by smearing a drop ofthe blood on the test paper, and after keeping it at room temperature for 10 min, placing a drop of the alkaline buffer on the paper, and then observing the color change under a UV-lamp. Both the routine and test paper methods are ap-plicable for testing the most common edible fish.

Alcoholic fermentation by ‘non‐fermentative’ yeasts

All type strains of 'non-fermentative' yeasts, available in the culture collection of the Centraalbureau voor Schimmelcultures, were reinvestigated for their capacity to ferment glucose in the classical Durham tube test. Although visible gas production was absent, nearly all strains produced significant amounts of ethanol under the test conditions. Under conditions of oxygen-limited growth, even strong alcoholic fermentation may occur in a number of yeasts hitherto considered as non-fermentative. Thus, shake-flask cultures of Hansenula nonfermentans and Candida silvae fermented more than half of the available sugar to ethanol. It is concluded that the taxonomic test for fermentation capacity, which relies on detection of gas formation in Durham tubes, is not reliable for a physiological classification of yeasts as fermentative and non-fermentative species.

Rapid method for detection, identification, and susceptibility testing of enteric pathogens.

Three hundred and seven colonies believed to be enteric pathogens were selected from primary plates of MacConkey, xylose desoxycholate, or salmonella-shigella agar for inoculation to lactose-sucrose broth, urea-41 motility medium, modified Andrade glucose broth with inverted Durham tube, pregrowth broth, triple sugar iron agar, lysine iron agar (LIA), and Christensen urea agar. The rapid screen consisted of interpreting the lactose-sucrose, urea-41 motility, and modified Andrade glucose broth gas reactions after 4 to 6 h at 35 degrees C. These rapid screening biochemicals plus LIA were incubated for 24 h if further interpretation was required. Reference biochemicals (triple sugar iron, LIA, and Christensen urea agars) were interpreted at 24 h. Of 307 isolates, 49 (16%) were reported as negative for enteric pathogens after 4 to 6 h because their biochemical profiles were not compatible with those for enteric pathogens. A total of 87 (28.3%) isolates produced biochemical profiles at 4 to 6 h that were presumptive for enteric pathogens. The 87 presumptive pathogens were inoculated into the AutoMicrobic system Gram-Negative General Susceptibility Card and the AutoMicrobic system Enterobacteriaceae-Plus Biochemical Card (AMS-EBC+) after 4 to 6 h of growth in pregrowth broth. Of these isolates, 63 were confirmed to be enteric pathogens, of which 61 (96.8%) were correctly identified by the AMS-EBC+. One isolate was identified as Shigella dysenteriae by AMS-EBC+ but confirmed as Shigella flexneri biotype 6 by a reference laboratory. The other isolate was identified as Arizona hinshawii by AMS-EBC+ but was confirmed as Salmonella enteritidis. Of the 307 isolates, 166 (54.1%) required further interpretation of the rapid screening biochemicals plus LIA at 24 h; 5 of these were detected as enteric pathogens. The same 68 enteric pathogens were detected by both the rapid method and the reference method. The results from the general susceptibility card agreed with agar diffusion results at 99.2%. One Salmonella enteritidis and four Shigella spp. showed minor discrepancies with tetracycline. No very major or major discrepancies were observed.

Degradation of organic acids by dairy lactic acid bacteria

One hundred and twelve different strains of lactic acid bacteria, belonging to the genera Leuconostoc, Streptococcus, and Lactobacillus, were examined for the ability to degrade 10 organic acids by detecting gas production, using the conventional Durham tube method. All the strains did not break down succinate, glutarate, 2-oxo-glutarate, and mucate. Malate, citrate, pyruvate, fumarate, tartrate, and gluconate were variably attacked. Streptococcus cremoiris AM2, ML8, and SK11 required glucose to produce gas from citrate, whereas Leuconostoc citrovorum and Streptococcus faecalis did not. Streptococcus cremoris differed from the other streptococci in not producing gas from gluconate. From all lactic acid bacteria examined, only Lactobacillus plantarum formed gas from tartarate. Determination of acetoin and diacetyl proved to be a more reliable evidence for assessing the degradation of pyruvate, compared with detection of gas production. Homofermentative lactobacilli and Leuconostoc citrovorum produced acetoin and diacetyl from pyruvate, whereas beta-bacteria did not, a character that would be of taxonomic value. Streptobacteria degraded pyruvate in the presence of glucose with lactate as the major product together with a mean acetate of 4.1%, ethanol 7.9%, acetoin 1.7%, and diacetyl 2.6% yield on a molar basis after 60 days at 30 degrees C. L. brevis produced acetate and lactate. Formation of diacetyl from pyruvate by lactic acid bacteria may play an important role in flavour development in fermenting dairy products, especially in cheese, where lactic acid bacteria usually predominate, and pyruvate is probably excreted in the breaking down of lactose and in the oxidative deamination of alanine by the accompanying microflora.

Evaluation of non-lactose-fermenting members of the Klebsiella-Enterobacter-Serratia division. I. Biochemical characteristics.

Use of selected biochemical media to identify and differentiate non-lactose-fermenting (NLF) members of the Klebsiella-Enterobacter-Serratia division is described. Media of particular usefulness in addition to routine tests are the DNA-raffinose plate, 1% glucose broth with Durham tube, and a combined ornithineindole-motility tube. By this method 330 isolates of Serratia and 142 of NLF Klebsiella-Enterobacter were identified. Most of the latter (83) were found to be Enterobacter species.

Palatinose Element of the Receptor of the Melezitose Locus in Saccharomyces

THE receptor1,2 of the melezitose (MZ) gene-system comprises at least five ‘elements’ corresponding to five α-glucosidic inductors of the enzyme melezitase, an α-glucosidase capable of splitting the five different α-glucosidic inductors of MZ, namely, turanose, maltose, sucrose, α-methyl-glucoside and melezitose3,4. The totipotent allele, which responds adaptively to all five substrates, is designated MZTMSGZ (abbreviated to TMSGZ). Partially recessive alleles, for example, TMSGz, TMSgz, TMsgz, Tmsgz, and the totally recessive allele, tmsgz, were obtained by ultra-violet irradiation5. (The small letters indicate specific inabilities of cultures to respond adaptively to the corresponding substrates.) The multiple alleles are inferred, on the receptor-hypothesis1,2, to reflect mutations (heritable changes in folding) of the proteinaceous receptor, a component of the gene controlling the capacity to respond to different inductors. It is of special interest to know if the synthetic α-glucoside, palatinose (α-glucosido-6-fructofuranose), can act as a substrate and an inductor of this particular enzyme. Emeis6 has already shown that Saccharomyces cultures carry genes controlling the fermentation of palatinose, but since there are other genes which split α-glucosides (and fructofuranosides), and since he did not have genetically identified MZ cultures with which to make specific tests, further analysis was necessary to implicate MZ. The totipotent TMSGZ cultures of the Lindegren Breeding Stock are able to ferment palatinose after 48 h, but specific partial mutants, for example, TMsgz and recessive tmsgz cultures, do not ferment palatinose when tested on EMB-agar and by the Durham tube technique (remained negative for five days)7. The fact that the TMSGZ cultures fermented palatinose suggested that palatinose might be one of the substrates (and possibly an inductor) by which the different alleles of the MZ locus may be distinguished from each other.

A FERMENTATION TUBE AND MEDIUM FOR FASTIDIOUS ANAEROBES.

A modified fermentation tube was devised by introducing a glass rod in the small inverted Durham tube to keep its mouth away from the bottom of the outer main tube, thus providing the maximum opportunity for gas collection. A medium containing autoclavable growth factors was developed, which, when added to the main tubes with a few particles of cooked meat, allowed growth of fastidious anaerobes. Each ingredient was shown to be free of any fermentable substance that might lead to gas formation. The fermentation tubes were incubated in an anaerobic jar.

Bacterial Oxidation of Lactose and Melibiose

DURING an investigation of bacteria isolated from blocks of whale meat, two distinct strains of paracolon bacteria (Blx, urease-positive; and C6, urease-negative) were obtained, the sugar metabolism of which was interesting. Tested in the normal way with Durham tubes, these organisms produced acid from lactose within 5–12 days at 30° C. and were accordingly recorded as late lactose fermenters. Tested by Hugh and Leifson's1 method, both strains fermented cellobiose, maltose, sucrose, trehalose, galactose, glucose, mannitol and sorbitol, producing acid and gas anaerobically within 48 hr. at 30° C. Lactose, melibiose and glycerol, however, were not fermented but oxidized, that is, acid was produced from these sugars within 48 hr. under aerobic conditions only.

Detection of gas production from glucose by heterofermentative lactic acid bacteria.

SUMMARY: Several modifications of the Durham tube method for detecting gas production have been tested with a variety of heterofermentative lactobacilli, some of which had been described in the literature as failing to produce gas from glucose by this method. Gas production in Durham tubes was detected consistently when the surface of a tomato juice broth medium containing 0·5% or 2·5% (w/v) glucose was sealed with liquid paraffin or with washed agar, but less consistently when the surface of the medium was unsealed. A minority of the strains examined grew weakly in a peptone + yeast extract medium containing glucose and produced very little titratable acidity or detectable gas. These produced large quantities of gas in the same medium when maltose replaced glucose.

Delayed fermentation of sucrose by certain haploid species of Saccharomyces.

Several haploid species ofSaccharomyces andSchiz. octosporus were shown to ferment sucrose in Durham tubes after a delay of 3 to 4 weeks. Detailed studies were done with a strain ofS. rouxii. The delayed fermentation of sucrose was not caused by mutationselection or by inducible enzyme formation, since young glucose grown cells after drying, freezing, aging or autolysis contained an active sucrase. Cells pretreated by drying or freezing fermented sucrose nearly as fast as glucose. After autolysis, the sucrase ofS. rouxii is only present in the cell debris and not in the autolysate. The use of a heavy inoculum in the van Iterson-Kluyver fermentometer resulted in a slow, but non-delayed fermentation. Variation in the pH or sucrose concentration had little effect on the delayed fermentation. It is suggested that after sufficient aging of the cells, the cell wall permeability undergoes a rather abrupt change, allowing the sucrose to come in contact with the sucrase of the cells.

RAPID MICROTECHNIQUES FOR THE FERMENTATION OF CARBOHYDRATES BY YEASTS

Two methods for the detection of fermentation by yeasts are described. Fifty yeast species, representing thirteen genera, have been examined and the results have been found to agree well with those obtained using Durham tubes. The new techniques depend upon the use of large inocula in comparatively small volumes of substrate and are so sensitive as sometimes to give positive results within 15 min. In addition, the small amount of medium required results in a considerable saving of materials with corresponding reduction in cost.

AN ANALYSIS OF THE PHENOMENON OF LONG–TERM ADAPTATION TO GALACTOSE BY SACCHAROMYCES

IT HAS LONG been known that Saccharomyces cerevisiae ferments galactose by an adaptive enzyme produced in the cell during the course of a few hours in the absence of cell division. Winge and Roberts (1948) have studied a culture of S. Chevalieri which ferments galactose by long-term adaptation which occurs after cells have grown from 3-14 days in a medium containing galactose. After the adaptation, the fermentation itself occurs at a rapid rate. They state that long-term adaptation of S. Chevalieri to galactose does not involve a shift in the population (mutation followed by selection) but is an adaptation of the entire original population. They consider the delay an indication of the time required by the cells to build up galactozymase to a sufficiently high concentration to make it demonstrable. They made hybrids between these two species and found a regular segregation of rapidly fermenting (1-2 days) to long-term adapting (3-14 days) clones from each ascus, provided the ascospores were isolated in beer wort, a medium containing considerable amounts of maltose, but lacking galactose. If the ascospores were isolated in galactose broth, all four spores from each ascus fermented galactose immediately (1-2 days). However, they used only asci in which all four spores germinated and the possibility exists that some nonutilizers of galactose may have failed to grow in maltose. They described a series of experiments in which they deadapted and readapted galactose fermenting cultures and denied the significance of mutation as a factor in this process. Extended genetical analyses bv Lindegren (1949) have established the allelism of a single pair of galactose genes (G/g) in Saccharomyces. One of these alleles (G) controls the rapid fermentation of galactose while cells carrying the other (g) do not effect detectable fermentation of galactose under the conditions of the Durham tube fermentation test. (Occasional irregularities in which an excess of fermenter (G G G g) or an excess of nonfermenter (g g g G) alleles were obtained from single asci have been interpreted as the result of conversion of alleles in the heterozygote.) We have discovered occasional cultures in our pedigrees which resemble the cultures of S. Chevalieri described by Winge and Roberts, by fermenting galactose after a long exposure involving growth; the present paper deals with the behavior of these unusual slow fermenters of galactose. We have concluded that this involves mutation from the g to the G allele followed by selection of the mutant G population in the presence of galactose.

The Fermentation of Trehalose by Yeasts and its Taxonomic Implications

SUMMARY: In the classification of yeasts it is customary to use an infusion of bakers’ yeast as the basal medium for fermentation tests. This extract frequently contains variable amounts of trehalose. A number of yeasts were observed to ferment yeast extract and trehalose. The fermentation of yeast extract is serious from a taxonomic point of view, since it gives the impression of positive fermentation of a sugar which actually may not be fermentable. Dilute yeast autolysate should be used as the basal fermentation medium since during autolysis trehalose is destroyed. The fermentation of yeast extract (without added sugar) is easily observed in Durham tubes by the collection of gas in the inserts, but when Einhorn fermentation tubes are used gas production is seldom apparent. One culture (N-18) isolated from spoiled apricots, and identified as Candida tropicalis showyed adaptive trehalose fermentation. The ability of various yeasts to ferment trehalose was investigated, using 133 cultures, representing twenty genera and seventy-three species. Sixteen species representing seven genera fermented yeast extract and trehalose. The fermentation of trehalose is worthy of consideration as a character for use in differentiating certain species of Candida, and perhaps other yeasts.

The Classification of Strains of Candida (Monilia) Isolated from Sputum

SummaryThe sputa of 693 sanatorium patients have been studied and 250 strains of the genus Candida isolated, an incidence of 36 per cent. Of these 205 (82 per cent) have been classified as C. albicans, 40 (16 per cent) as C. tropicalis, 2 (0.8 per cent) as C. Krusei, and 3 (1.2 per cent) strains remain unclassified. Fifteen cultures from other laboratories have also been compared using our method of study. By inoculating a plate containing malt agar and modified Durham tubes containing sucrose and maltose the great majority of strains isolated from sputum can be classified. The procedure is, therefore, simple and the media are easily prepared. Other methods should be resorted to if this plan fails. The generic term Candida has been substituted for Monilia. Different names for some of the species have also been used.

Amebiasis and Bacillary Dysentery in the Los Angeles County Hospital 1929–1935 1

Summary 1. Dysentery in Southern California is caused primarily by three types of microörganisms, Shigella paradysenteriae, Salmonella morgani, together with several other species of Salmonella and Endamoeba histolytica. 2. Acute bacillary dysentery is encountered much more frequently than acute amebic dysentery. These two types may often be differentiated by microscopic examination of the stool, the bacterial type usually showing a prolific cellular exudate with a preponderance of polymorphonuclear leucocytes and the amebic type showing a scanty exudate made up almost entirely of macrophage cells in addition to the trophozoites of amebae. 3. Chronic amebiasis is proportionately more common than chronic bacillary dysentery. The latter, however, occurs more frequently than many suspect and should be suspected as being associated with Shigella or Salmonella rather than with Bargen's Streptococcus. 4. Carriers of E. histolytica, Shigella and Salmonella have been observed, the bacterial types being somewhat less common than the amebic. 5. Recently an outbreak of bacillary dysentery occurred which was caused by the Shigella paradysenteriae “Sonné.” 6. Although the agglutination reactions are diagnostic, the lactose fermentation is much slower and less regular in occurrence than such reactions commonly reported for “Sonné” and also less regular than those produced by five other strains of “Sonné” with which they were compared. 7. Fermentation occurs first under anaerobic conditions, either in the Durham tubes of lactose broth medium or at the bottom of a lactose agar slant. Fermentation, in media in which the Durham tube is not present, occurs more frequently in nutrient broth medium, than in dibasic phosphate peptone medium. 8. Rough and smooth colonies and “satellite” colonies may give rise either to rapid or to slow lactose fermentation. 9. These variations from the usual descriptions given for the “Sonné” type of organism should be considered in the laboratory diagnosis of dysenteric bacteria for certain non-lactose fermenting strains which fail to agglutinate with the common antisera, may be found to belong to the “Sonné” type.

Precipitin and Fermentation Reactions of the Moniliae

In the course of the study of smears from cases of bronchial asthma and routine cultures taken from cases of ringworm infection of the feet, numerous yeast-like organisms were encountered, especially those of the monilia group. Since fermentation reactions and serological tests proved to form a very important basis for the identification of the moniliae, as well as other yeasts, further studies have been made of these 2 characteristics. Previous investigators, in the study of fermentation reactions, have made their observations on extract broth plus 1% fermentable sugars with Andrade's solution as an indicator, and Durham tubes for determination of gas. Because of the differences in the results of these authors, some method seemed necessary to investigate this test further. Pork infusion fermented broth (the carbohydrates in the meat infusion were removed by fermentation with B. coli before the peptone was added) was used with addition of the various carbohydrates. The medium was inoculated with monilia and incubated at 37°C. The pH of the inoculated media was followed every 2 or 3 days for 20 to 40 days by the colorimetric method described by Brown. Eight or 10 drops of the uninoculated media gave distinctly positive reactions for sugar with Benedict's solution. Those cultures found to give a negative chemical test for sugar after a period of incubation were considered to have fermented the carbohydrates. The hydrogen-ion concentration of the cultures of moniliae in 2% sugar broth over a period of 40 days showed a wide divergence in the amount of acidity produced in the fermenting process. Several strains, although showing little if any acidity, fermented the sugar as shown by its disappearance indicated by the negative Benedict's test. Taking the disappearance of the sugars as a basis for division, the moniliae fall into 3 groups: A first group, M. albicans, M. Candida and M. psilosis, utilizes completely sucrose, dextrose, maltose, xylose, levulose and galactose, but does not attack inulin. A second group including M. parapsilosis and M. type 1 (Stovall), utilizes 4 of the above sugars not fermenting maltose or inulin and rarely xylose. A third group includes M. krusei which does not utilize xylose, sucrose, maltose, galactose or inulin.

Physiology of Shigella paradysenteriae var. Sonne

The dissimilation of lactose and sucrose by Shigella paradysenteriae var. Sonne has been studied to gain additional knowledge of the phenomenon known as “delayed” or “slow” fermentation. The culture (A.T.C. 31) shows in lactose and sucrose the delayed fermentation characteristic of the Sonne type. In Durham fermentation tubes about 4 days are required for acid formation in lactose broth and about 7 days for sucrose. Colonies on lactose or sucrose agar plates are alkaline or neutral but after a few days develop small secondary colonies as papillae which become acid. If a papilla on sucrose or lactose agar is suspended in sterile broth or saline and plated on agar containing the same sugar, 2 types of colonies appear. One type is acid and does not form papillae even after long incubation, the other type is like the original, alkaline and develops acid papillae. Sugar broth inoculated with the acid type colony is promptly acidified, whereas the alkaline type causes the characteristic delayed fermentation. Attempts to decrease the time for acid formation in the slow culture by transferring once or twice daily to fresh medium with the same sugar have failed, though this procedure has been carried through 60 transfers. One may easily obtain the rapid variant, however, by plating out a culture which has developed acid, the types of colonies developing on the plates being of 2 types, acid and alkaline, as when the papillae from plate cultures are used. In order to study the fermentation of lactose and sucrose in more detail than was possible using Durham tubes, flasks have been used, and examination made at least once daily to determine pH, titrable acidity, oxidation-reduction potential, sugar utilized, total population and per cent of population composed of the rapid fermenting variant.

A Study of Coliform Organisms in samples of “Certified Milk.”

The experiments herein described show that:(1) Of the 268 coliform organisms isolated from 32 samples of certified milk, 65.7 per cent, fall within the true B. coli group and 18–6 per cent, in groups X and Y of Stewart.(2) 54–1 per cent. of the organisms, i.e. those of the B.faecalis alkaligenes group and those in subgroups 1, 2 and 3 of MacConkey which fermented lactose, gave a negative V. and P. and a positive M.R. reaction, can be assumed to be of faecal origin.(3) 45–9 per cent., namely those organisms of the B. proteus group, those of subgroup 4 of the B. coli group and those of groups X and Y are of the type generally found in soil and water.(4) All of the 32 samples examined contained coliform organisms in a dilution of 1 in 10 as shown by bile salt agar plates. None of these samples, however, gave a positive reaction with the presumptive test in two out of three tubes of 1/10 c.c. dilution. The presumptive test cannot, therefore, be relied upon to give a true indication of the presence of coliform organisms in milk. The inaccuracy of the test may be due in part to the structure of the ordinary Durham's tube which fails to ensure the collection of the gas produced.(5) Although the majority of the coliform organisms present in milk ferment lactose other members of the group which are not lactose fermenters occur and consequently their presence is not demonstrated by the presumptivetest.