THE ADAPTATION OF FUNGI TO FUNGICIDES: ADAPTATION TO COPPER AND MERCURY SALTS

The susceptibility of Botrytis cinerea to copper sulphate in liquid media increased when the volume, and therefore the depth, of the medium in culture bottles exceeded certain values; when the volume was 40 ml. the maximum concentration allowing growth was 300 p.p.m.
By growing mycelium in media containing progressively higher concentrations of copper sulphate a strain was produced which grew at a concentration of 750 p.p.m.
In high concentrations of copper sulphate growth always started at the edge of the liquid, and inocula grew only if they were placed in this position.
In germination tests spores from the resistant strain were more resistant to copper sulphate than were spores of the parent strain.
The resistance of mycelium, and to a lesser extent of spores, was retained after growth of the resistant strain for six months in fungicide-free media.
Spore and mycelial inocula grew in much higher concentrations of copper sulphate when nutrient media were solidified with agar.
The strain resistant in liquid media was no more resistant than the parent strain on agar media.
The resistance of the fungus was not increased after growth for long periods on agar containing high concentrations of copper sulphate. The resistance of the strain resistant in liquid media was not lost after growth on agar media for 3 months.
Attempts to produce strains more resistant than the parent to mercuric chloride were unsuccessful.
The results obtained with phenyl-mercuric acetate were essentially similar to those obtained with copper sulphate, but relatively much more resistant strains were produced.     

Comparison of most probable number and pour plate procedures for isolation and enumeration of sulphite-reducing Clostridium spores and Group D faecal streptococci from oysters

A comparative study of methods to enumerate sulphite-reducing Clostridium spores and Group D faecal streptococci in oysters demonstrated that pour plate solid agar techniques gave higher counts than liquid broth most probable number procedures. Reinforced clostridial broth with supplements to detect sulphite reduction was compared with pour plates of egg yolk-free tryptose sulphite cycloserine agar incubated at 37°C for 24 h. Azide dextrose broth was compared with pour plates using Slanetz and Bartley (SB) agar or KF-streptococcus agar at 37°C. Most probable number procedures used for both groups of organisms gave excessive numbers of improbable tube combinations. For enumeration of Group D faecal streptococci, a pour plate technique using SB agar incubated at 37°C for 48 h is recommended.     

THE SELECTIVE ACTION OF THALLOUS ACETATE FOR LACTOBACILLI

SUMMARY: The effect of thallous acetate has been examined on the growth of 23 strains of lactobacilli representing 13 different species. All strains grew satisfactorily in both broth and agar media containing 0.1% of thallous acetate. Of the 18 other organisms also examined the growth of the streptococci was unsuppressed, staphylococci, a micrococcus, B. licheniformis and Pr. mirabilis were partially inhibited and strains of Bact. coli, B. cereus, Chr. prodigiosum and Ps. fluorescens were almost completely inhibited by that concentration. By its use a valuable selective medium for isolating lactobacilli from natural habitats of mixed flora was obtained, other organisms, with the exception of streptococci, being almost completely eliminated.     

THE DETERMINATION OF THE COLI-AEROGENES CONTENT OF MILK AND DAIRY EQUIPMENT BY PLATING ON VIOLET RED BILE AGAR INCUBATED AT 30°

SUMMARY: Violet red bile agar (VRB) incubated at 30° for 20–24 hr was as good an indicator of coli-aerogenes bacteria in milk as MacConkey's broth. A high proportion (82%) of the large, deep red colonies considered to be formed by coli-aerogenes bacteria were confirmed as such. A British brand of dehydrated VRB agar was as suitable as an American brand of this medium for determining the coli-aerogenes content of milk. All the strains of typical coli-aerogenes bacteria tested formed red colonies. In a small proportion of cases the diameter was less than 0·5 mm. The only other milk bacteria which formed colonies resembling those of coli-aerogenes organisms were some acid forming strains of Gram-negative rods. Coli-aerogenes bacteria, determined on VRB agar at 30°, generally constituted only a small proportion of the microflora of fresh raw milk and of farm dairy equipment.     

A COMPARISON OF 30° AND 37° AS INCUBATION TEMPERATURES IN THE PRESUMPTIVE COLIAEROGENES TEST FOR RAW AND PASTEURIZED MILK

SUMMARY: Incubation at 30° and 37° for the presumptive coli-aerogenes test for raw and pasterurized milk has been investigated. There were more positives at the lower temperature and it is suggested that in this test, incubation at 30° might provide a much better guide to the hygienic quality of both raw and pasterurized milk. The ability of the coli-aerogenes bacteria studied to ferment lactose in MacConkey's broth at 30° but not at 37° was found to be a stable factor which was unchanged by prolonged storage on agar slopes at room temperature or on continued incubation in MacConkey's broth at temperatures above the optimum for lactose fermentation.     

THE ENUMERATION OF FAECAL STREPTOCOCCI IN FOODS, USING PACKER'S CRYSTAL VIOLET SODIUM AZIDE BLOOD AGAR

SUMMARY: Packer's crystal violet sodium azide blood agar (Packer, 1943) used in poured plates at 36·1°, gave satisfactory recovery of pure cultures of Lancefield group D streptococci and completely inhibited the growth of 11 other species of aerobic and anaerobic food bacteria, including Strep. lactis (5 strains). Later, however, one group N streptococcus was obtained which did grow in Packer's agar at 36·1°. To eliminate this organism the incubation temperature had to be increased to 39·5°, using agar strips (Stirling et al. 1950) incubated in a water bath to secure strict temperature control. Under these conditions the recovery of typical group D streptococci was never consistently below 50% of the count in tryptone dextrose yeast extract agar at 31·1°.     

COMPARISONS OF DIFFERENT MEDIA FOR COUNTING SUGAR TOLERANT YEASTS IN CONCENTRATED ORANGE JUICE

SUMMARY: To select and count the sugar tolerant yeasts which ferment sixfold concentrated orange juice, a high sugar agar medium was developed which contains 50% of glucose, 1% of citric acid and 1% of Tryptone; it is incubated for 4–5 days at 25°.
The medium has disadvantages: it is troublesome to prepare, and colonies grow slowly and are translucent. These properties result directly' from the high sugar concentration, on which the selective action of the medium depends.
Counts on this medium have been compared with those on potato dextrose or nutrient dextrose agars (with 2% and 1% of glucose respectively), with yeasts isolated from fermenting concentrate, in pure culture, and under various practical conditions. As a rule, the counts were virtually the same on the different media; nutrient dextrose agar occasionally failed to record small numbers of these yeasts. If the two low sugar media were acidified to pH 3·5 the counts were reduced.
Potato dextrose agar recorded, besides the above yeasts, sugar intolerant yeasts entering from dirty machines or through bad canning practice: nutrient dextrose agar recorded bacteria in addition. The difference between parallel plating on these media and on the high sugar medium thus yielded useful information about sources of casual contamination.
It is suggested that the above would also be largely applicable to other sugar-rich concentrates of not less than 50° Brix.     

Use of cereals as basal medium for the formulation of alternative culture media for fungi

Summary The feasibility of developing alternative media to different culture media particularly potato dextrose agar was assessed using local cereal species as the basal media. Three cereal meal extracts – corn, sorghum and millet – were prepared, using them as substitute for the potato in potato dextrose agar. Potato dextrose agar (PDA) was the standard set up with which the performances of the formulated media were compared. Eight genera of fungi (Aspergillus niger, Fusarium moniliforme, Penicillium sp., Cercospora sp., Curvularia palescens, Botryodiplopodia sp., Rhizopus sp. and Rhodotorula rubra) were isolated and pure cultures of each species aseptically inoculated onto the three different formulated media including PDA and allowed to grow. Their growths were measured at 24, 48, 72, and 96 h after inoculation, using diameter of growth as an index. The set up was repeated thrice for each species on the three formulated media and the control (PDA). Growth of all the fungal species were observed to be about the same or sometimes better in the formulated media relative to those on the standard set up, except for Rhodotorula rubra. The radius of growth of F. moniliformehad an average of 15 + 0.58 mm on corn-dextrose agar relative to 12 mm on PDA at 96 h while Cercospora sp. measured 30 + 0.58 mm on millet-meal dextrose agar relative to 37 + 1.16 mm at 48 h. Botryodiplopodia sp. grew through the whole diameter of the plate (covering the total length of the radius of 45 mm) in both sorghum-meal and PDA at 96 h.     

新生隐球菌交配型分析试验方法和培养基的研究

比较观察了Hayagar(HA)、Matingagar (MA)、Haycubeagar (HCA)、稻壳琼脂(RSA)、富营养Hayagar(IHA)以及沙保弱培养基 (SDA) 6种培养基的新生隐球菌交配试验效果。按 2周内产生阳性结果排列培养基的次序是HCA(95% )、RSA(95% )、HA(86% )、MA(38% )、SDA(38% )和IHA(2 4 % )。采用点状可以替代划线接种方法用于处理大批量标本并节省材料。此外 ,先将诱导菌株和受试菌株混合预培养可以使原方法不能交配的菌株产生阳性结果     

A System for Detecting Salmonellae in Meat and Meat Products

Leifson's selenite F broth was more selective for salmonellae when incubated at 43° instead of the traditional 37°. Different selective agar media produced different numbers of colonies from similar inocula of salmonella cells, but Difco brilliant green agar consistently gave the highest recoveries when tested in this way. Combined with 43° selenite broth enrichment it provided a useful system for isolating salmonellae from foods. In a short comparative test this system compared favourably with more classical techniques employing enrichment of each sample at 37° in two different enrichment broths, followed by streaking on two selective agars.     

STUDIES OF THE DETERMINATION OF THE BACTERIUM COLI TYPE I CONTENT OF FARM WATER SUPPLIES

SUMMARY: A – comparison of the suitability of brilliant green bile broth and MacConkey's broth at 44° for the detection of Bacterium coli type I in farm water supplies, showed that 83.1% of the samples had no difference in the number of positive tubes at 44°, and only 5 samples (1.7%) had a significantly higher number of positive tubes in MacConkey's broth.
Of 707 strains of coli-aerogenes bacteria isolated from 44° positive tubes of both media, 94.5% were Bact. coli type I. Strains of Bact. coli type II and Bact. aerogenes type I which were 44° positive constituted 3.7% and 0.4% respectively, all of which were indole negative at 44°. In addition there were 10 strains (1.4%) of 44° positive Intermediate type II, 9 of which were indole positive at 44°.
An appreciable number (6.6%) of Bact. coli type I strains failed to give a positive indole reaction in 24 hr at 44°.     

SOME BIOCHEMICAL CHARACTERISTICS OF FRESHLY ISOLATED STRAINS OF THE GENUS SERRATIA

SUMMARY: Many of 110 strains of Serratia , isolated from soil, water, milk and dairy equipment, were biochemically closely related to the coli-aerogenes bacteria. Acid and gas was formed from glucose in 14 days at 30° by 53% and from lactose and MacConkey's broth by about 40%. All except one strain gave——++ IMViC reactions.
An inverse relationship was observed between depth of pigmentation and carbohydrate fermentation. Complete loss of pigment in mutant strains was not uncommon, and was associated with loss of proteolytic properties and increase of saccharolytic activity.
The majority of the strains had psychrophilic characteristics: 75% grew at 3–5°. Most strains showed moderate growth at 37°, but only 7 formed red pigment at that temperature.
All strains resembled Serratia marcescens in morphology, containing minute coccoid rods smaller than those of coli-aerogenes bacteria.     

A stable L-form of the fish pathogen Aeromonas salmonicida

A stable L-form of Aeromonas salmonicida , which resulted from induction with benzylpenicillin, grew on brain heart infusion agar at 0–5°C. The L-form was stored successfully for 10 months in phosphate-buffered saline supplemented with 150 g l^-1 glycerol at -70°C. Reversion of the L-form to parental-type walled cells was achieved by transfer to brain heart infusion broth with incubation at 25°C.     

Comparison of the Effects of Liquid Medium Repair and the Incorporation of Catalase in MacConkey Type Media on the Recovery of Enterobacteriaceae Sublethally Stressed by Freezing

Nine pure cultures of species of Enterobacteriaceae were stressed by rapid freezing in tryptone soya broth (TSB) to — 22°C and subsequent storage at that temperature for 7 d. About one to two log cycles kill and at least one additional log cycle sublethal impairment was achieved. Numbers of colonies of these cultures in poured plates of violet red bile glucose (VRBG) agar, with 67 u/ml of catalase added at 47°C, were only slightly higher than those in plain VRBG, both incubated overnight at 30°C. Two hours incubation of TSB suspensions at 17–25° C resulted in almost complete restoration of the ability of cells to develop colonies in VRBG, without, however, leading to any significant multiplication.
Similar experiments with 32 samples of frozen minced meat, 27 samples of frozen surface water, 18 of frozen chicken liver and 14 of fresh sausage substantiated the results obtained in the studies on pure cultures.
In the experiments with the nine pure cultures the influence of the nutrient composition of the solid enumeration media: 'minimal' agar, TSB agar (TSBA) and Mueller-Hinton agar with Polyvitex nutrient supplement (MHA), on the recovery of Enterobacteriaceae stressed by freezing was also studied. Colony numbers in TSBA and MHA were virtually identical. The glucose mineral salts medium led to lower recovery, indicating that so-called 'minimal medium recovery' of stressed bacterial populations is not a common phenomenon.     

Enumeration of intestinal enterococci and interfering organisms with Slanetz-Bartley agar, KF streptococcus agar and the MUST method

The recovery of intestinal species of enterococci and streptococci and potentially interfering nonfaecal species was measured on KF streptococcus agar, Slanetz-Bartley agar and in a medium based on 4-methylumbelliferyl-β- d -glucoside (MUST method) using pure cultures. Both of the solid media yielded high recoveries of the target species. Their selectivity was better at elevated incubation temperature but nonfaecal Enterococcus and Staphylococcus species were not eliminated even at the elevated temperature. The MUST method tended to give slightly lower recoveries than the agar cultivation methods with some target species at 44°C but recoveries were better at 41°C.     

利用DGGE评价不同培养基回收番茄根际细菌类群的能力

用营养肉汤、YG、根系分泌物、土壤浸渍液4种培养基从番茄根际分离培养细菌,并结合变性梯度凝胶电泳(DGGE)技术,对4种培养基回收番茄根际细菌种群的能力进行了比较研究。结果表明,不同培养基和培养温度,回收到的细菌种群有一定差异;低营养浓度的YG培养基在较低的培养温度20℃下进行较长时间的培养,比高营养浓度营养肉汤培养基产生更多、更具代表性的细菌;以根系分泌物为基础的培养基从番茄根际回收到的优势菌群最多。该研究初步建立了用DGGE技术对不同培养基回收分离细菌种群能力进行评价的方法。     

THE PROPERTIES OF TOMATO ASPERMY VIRUS AND ITS RELATIONSHIP WITH CUCUMBER MOSAIC VIRUS*

Chrysanthemum plants infected with tomato aspermy virus (TAV) produce severely distorted and discoloured flowers but show only slight leaf mottle.
TAV infected twenty-five of forty-five species (belonging to seventeen genera) tested and was transmitted by the aphid species Aulacorthum solarti, Macrosiphoniella sanborni and Myzus persicae .
Sap from infected tobacco leaves lost infectivity when diluted more than 1 in 10,000, when heated for 10 min. at above 65°C. and when stored for more than 42 hr. at 16–18°C.
Partial protection was obtained between TAV and two strains of cucumber mosaic virus. Evidence was obtained that this was true protection between related viruses and serological tests confirmed the view that TAV is a strain of cucumber mosaic virus. Evidence was obtained that this was true protection between related viruses and serological tests confirmed the view that TAV is a strain of cucumber mosaic virus.     

Formation and Germination of Septoria tritici Secondary Conidia as Affected by Environmental Factors

The effects of medium, isolate, temperature, light and pH on the formation and germination of Septoria tritici secondary conidia were tested. Of the six media tested, the malt–yeast extract agar was the best and generated 1.82 × 10⁹ conidia/plate. The ten isolates tested showed different ability of conidia production. Darkness significantly reduced conidial formation and enhanced the transition of intermediates. The conidial germination and germ tube growth was strongly inhibited at 30°C. The suitable pH for conidial budding in malt–yeast broth (MYB) was between 5 and 9. At pH 2, 10 and 11, almost no new conidia were formed. The number of conidia reached 1.27 × 10⁸ conidia/ml after 7 days in MYB, significantly more than that in potato dextrose broth, wheat leaf extract and H₂O.     

Effect of growth conditions and storage on the specific activity of β-lactamases of Bacteroides spp.

The influences of growth conditions and cold storage on the specific activity of β-lactamases of four strains of Bacteroides spp. was studied. Interbatch variation was observed in extracts prepared in an identical way on separate occasions but less variation was observed in extracts prepared from bacteria grown on Brain Heart Infusion agar supplemented with yeast extract, haemin and menadione, than in similar extracts of bacteria grown in broth or on other solid media. The loss of enzyme activity seen during the stationary phase of growth of some strains in broth was minimal during incubation for 48 h on agar. Storage of enzyme extracts at 4°C was associated with loss of enzyme activity, but activity was retained during storage at—70°C for up to 32 days. Freezing and thawing had little effect on enzyme activity.     

THE ENUMERATION OF LACTOBACILLI ON GRASS AND IN SILAGE

SUMMARY: For the enumeration of lactobacilli on grass and in silage the following medium has shown promise: peptone, meat extract and glucose, 10 g. each; tomato extract, 200 ml.; yeast autolysate, 50 ml.; Tween 80, 0.5 ml.; agar, 15 g., in a final volume of 1 1. and containing acetic acid/sodium acetate buffer in 0.2M concentration; pH 5–4. The medium was adjusted to pH 5–4 before sterilization and the requisite amount of concentrated pH 5.4 acetate buffer added just before plating. Double laver plates were used.
The only other silage organisms which in this medium formed colonies comparable in size with those of lactobacilli were heterofermentative streptococci and a micrococcus.