Durham tube

Aluminum block Durham tube 96 Deep well B 7 mm, L 40 mm microplale... 

Liquid media may also be used for testing oxidative/fermentative reactions (Hugh and Leifson s O/F test), and if this is done then a small inverted test tube known as a Durham tube is added. This allows the reaction to be examined for the formation of gas as well as acid. Tubes are again prepared in duplicate, and sterile liquid paraffin or mineral oil is again added to one tube to accentuate the anaerobic reaction. A few organisms may produce an alkaline reaction due to the conversion of peptones in the medium to amines. 

The medium may be solid or liquid. In the case of liquid medium, a Durham tube may be added in order to detect any gas which may be formed. The difference between acid, and acid plus gas may be of diagnostic value. 

To prepare the single-strength lactose-peptone broth, the nutrient broth described above is diluted with the same volume of demineralized water prior to the addition of the bromocresol purple indicator. The pH is then set and the indicator solution added. Test-tubes are then each filled with 10 ml of this solution a Durham tube is added to each test tube and the... 

Durham tubes are approx. - 5 cm long, similar to test tubes, with a diameter of 6 - 8 mm. One Durham tube is put into each test tube with its opening pointing downwards. During sterilization, the air in the Durham test tube escapes so that it is completely filled with liquid after the sterilization process. If the inoculated solution forms gas later during incubation, the gas collects in the Durham test tube. 

Since only the "single-strength" medium is used, the double-strength medium must first be diluted with the same volume of demineralized water. 3 ml of the diluted medium is poured into test tubes. After the addition of Durham tubes, this is sterilized in an autoclave at 121 C for 20 minutes. 

Sterile lactose-peptone broth in suitable vessels, double-strength for 100 ml and 10 ml water (broth I), single-strength for 1 ml and 0.1 ml water (broth II) with Durham tubes (see Recipe 3)... 

Among the physiological tests utilized in identification is the ability to ferment carbohydrates. This property can be visualized by the formation and, subsequently, collection of gas in inverted Durham tubes (see Fig. B-1). The medium uses 0.5% (wt/vol) yeast extract broth as a basal medium into which the carbon source of interest is incorporated. The inverted Durham tube is then placed into the medium, the tube capped, and then autoclaved. It is not necessary to attempt to remove the air bubble (s) in Durham vials. During the autoclave cycle, the gas will be displaced. 

Fig. B-1. Fermentation broth. Durham tube for detection of gas production.

Some sugars (glucose and galactose) can be autoclaved. These are independently incorporated into the yeast extract (YE) broth before autoclaving. Others must be filter sterilized and are added, after autoclaving, to the YE broth/Durham tube setup (see section 3.5.4). 

For sugars that can be autoclaved (e.g., glucose and galactose), prepare as 2% w/v solutions in the yeast extract broth and transfer 10mL to capped 18 X 150 mm test tubes. Insert a Durham tube open-side down into each tube, loosely replace the cap, and autoclave at 121 C/250 F for 15 min. 

Method A is from Edwards et al. (1991), a protocol originally modified from Gibson and Abdel-Malek (1945) and Garvie (1984) while method B was described by Pilone et al. (1991). The presence of gas is indicated by the elevation of the agar plug inside of the tube (method A) or by gas trapped in the Durham tube (method B). If no gas is apparently produced using method A, it may be necessary to sonicate the tubes to force gas to coalesce into visible bubbles. 

Place 9 mL into sterilized 16 X 150mm test tubes containing an inverted Durham tube, cap, and autoclave at 121°C/250°F for 15 min. 

As an alternative to using Durham tubes, the inoculated HFA broth can be overlaid with approximately 1 cm of molten vaspar (mixture of 1 part petroleum jelly -I- 6 parts paraffin) to assist in visualizing gas formation. 

Durham tube A small test tube (9.5 X 50mm) placed inverted in a liquid broth and used to detect COj formation by physically trapping the gas. 

Brilliant green lactose bile broth (for total coliforms) 35°C-37°C + 0.5°C for 24- 8 h Gas production in Durham tubes [2,67]... 

Lauryl tryptose mannitol broth with tryptophan (presumptive for E. coli) Completed (Optional) Tests 44 C. 5°C for 24r-48 h Gas production in Durham tube and formation of a red ring after addition of Kovacs reagent [67]... 

For the presumptive test for total coliforms, lauryl tryptose broth (LTB) growth medium is used alor g with fermentation tubes with inverted vials (Durham tubes) for gas production. As an alternative to the use of Durham tubes, 0.01 g/L of bromcresol purple is added to the medium and used as an indicator. Serial dilutions of the water sample to be tested are made and inoculated into LTB growth media. Samples are then incubated at 35°C for 24 h and for an additional 24 h if no growth was observed at the end of the first 24 h. If coliform bacteria are present in the samples, growth (turbidity), gas bubbles, or acid will be produced in the tubes as a result of fermentation of lactose. The number of gas positive tubes or tubes in which acid reaction occurred is used to calculate the MPN of bacteria in the samples [2]. A 10-tube MPN is used to test bottled water samples whereas the 5-tube MPN is used to test freshwater, marine waters, and shellfish. 

Confirmation test for E. coli To confirm the presence of E. coli in total coliform positive samples, add MUG (4-methylumbelliferyl-(3-D-glucoronide) to EC growth medium at a concentration of 50 JLg/mL. MUG is a substance cleaved off of E. coli cell that contains the enz)me, (3-glucuronidase. This test is based on the cleavage of MUG to free methylumbelliferyl moiety, which fluoresces a blue color when irradiated with UV radiation. Before use, the EC medium should be sterilized the pH after sterilization should be 6.9 + 0.2. It is important to test the EC medium for fluorescence before use. Inoculate EC tubes from positive BGLBB tubes and incubate tubes in a water bath at 44.5°C for 22-26 h. The inverted Durham tube should be omitted. A positive reaction for E. coli is indicated by the presence of blue fluorescence. A tube inoculated with a known positive culture and a negative culture should be included for each batch to be tested for reference purposes in order to eliminate false positives. 

Lactose peptone water 35°C-37°C 0.5°C Gas formation in Durham tubes [70]... 

Dissolve 10 g of bacteriological peptone, 500 g of glucose, 1 gof sodium acid phosphate and 1 g of citric acid in water and make up to 1 litre. Distribute in about 20-ml amounts in tubes or bottles, each containing a Durham tube (for detecting the evolution of gas) and sterilise at 115° (10 lb steam pressure) for twenty minutes. 

Figure 1. An illustration of the Durham tube method. A Durham tube consists of a smaller test tube inverted inside a larger tube, both of which are sterile. To test for the presence of intracellular fermentable carbohydrate the larger tube is three-quarters filled with a suspension of cells, the smaller tube is inserted into the larger tube, and the cap is screwed tight The assembly is then inverted to fill the enclosed smaller tube with the suspension, righted, and incubated at an appropriate temperature (30 C is good for yeasts) as shown for tube A. If fermentable carbohydrates are present in the suspended cells, gas will accumulate in the top of the inverted smaller tube, causing it to float inside the larger tube, as shown for tube B...

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