|Year : 2013 | Volume
| Issue : 2 | Page : 173-176
Anaerobic culture on growth efficient bi-layered culture plate in a modified candle jar using a rapid and slow combustion system
PK Maiti, J Haldar, P Mukherjee, R Dey
Department of Microbiology, Institute of Post Graduate Medical Education and Research, AJC Bose Road, Kolkata, India
|Date of Submission||21-Aug-2012|
|Date of Acceptance||13-Nov-2012|
|Date of Web Publication||19-Jul-2013|
P K Maiti
Department of Microbiology, Institute of Post Graduate Medical Education and Research, AJC Bose Road, Kolkata
Source of Support: None, Conflict of Interest: None
Success for maximum isolation of anaerobes depends on maintaining critically low oxygen levels throughout and growth in a reduced medium with exclusion of inhibitory substances. Hence a dual system was used equipped with candle combustion for instant exhaustion of major part of oxygen from a sealed jar, along with acidified steel wool for residual oxygen purging. For inhibitory substances removal, test anaerobes were grown on anaerobic medium layered on buffer charcoal agar bed. After 48 hours incubation average colony sizes were compared with that of growths in conventional Gas-Pak system. Better growths were noted in the innovative system.
Keywords: Anaerobiosis, bacterial culture, candle-jar, combustion
|How to cite this article:|
Maiti P K, Haldar J, Mukherjee P, Dey R. Anaerobic culture on growth efficient bi-layered culture plate in a modified candle jar using a rapid and slow combustion system. Indian J Med Microbiol 2013;31:173-6
|How to cite this URL:|
Maiti P K, Haldar J, Mukherjee P, Dey R. Anaerobic culture on growth efficient bi-layered culture plate in a modified candle jar using a rapid and slow combustion system. Indian J Med Microbiol [serial online] 2013 [cited 2020 Dec 1];31:173-6. Available from: https://www.ijmm.org/text.asp?2013/31/2/173/115218
| ~ Introduction|| |
GasPak system is the most acceptable method for small-scale laboratories. It is based on self contained hydrogen-carbon-dioxide generator with a cold catalyst for oxygen reduction.  However, it is a costly, single use technique, and takes about 90 minutes to attain a critical low level of oxygen.  So bacterial growth is compromised in first few generations, which may be a factor for poor isolation rate. Alternatively, a candle jar technique can be applied for quick reduction of major bulk of oxygen and then residual 1-2% oxygen can be slowly reduced by applying a plate containing reducing chemicals.
| ~ Materials and Methods|| |
Commercially available, transparent, heat tolerant, gas-impermeable, hard, 1000 ml, 120 mm diameter, 150 mm high cylindrical poly-carbonate jar (Tarson, India), with screw-on air-tight lid was used. Each jar was checked for leaks.
For studying the extent of oxygen utilization by candle combustion, one vertically fixed lighted white-wax candle was firmly placed on center of a large Petri-plate almost filled with water. One measuring cylinder was gently inverted vertically over the candle. Within a moment the flame extinguished and water level inside cylinder elevated to fill the vacuum created after consumption of oxygen (O 2 ) and a little carbon-dioxide (CO 2 ) generation. This helped for a rough calculation of O 2 consumption following rapid combustion, supposing total O 2 in the air was 21% and generated CO 2 was 4%.
The oxygen reduction level in candle jar system was assessed by modified methylene blue indicator.  One such indicator containing tube was placed in test jar with lighted candle and also in another jar containing acidified steel wool along with a lighted candle. Jar lids were closed promptly. The indicator turned colorless and remained so for 7 days in latter case while it remained blue in ordinary candle jar. This indicated that oxygen was reduced to less than 0.05 atmospheres in modified candle jar system.
The basal medium for conventional anaerobic culture was Brucella More Details Agar (Hi-Media, India), which was supplemented with 1 μg/ml menadione, 5 μg/ml hemin, and 5% sheep blood. For preparing bi-layered test medium, 1% activated charcoal (Hi-Media, India) with phosphate buffer (PH 7.4) was added with Brucella Agar base. After solidification of this bottom layer, a layer of supplemented Brucella Agar was dispensed. Both types of media were prereduced by incubating in an anaerobic chamber for 48 hours.
The following quality control strains (MicroBiologics, USA) were included for observing growth characteristics in conventional and test anaerobic systems: Bacteroides fragilis ATCC 25285, Clostridium perfringens ATCC 13124, Clostridium sporogenes ATCC 11437. Four anaerobes isolated from our laboratory (one each) were also tested: Clostridium tetani, Peptostreptococcus anaerobius, Veillonella parvula, and Porphyromonas asaccharolytica.
Inoculation and incubation
Five grams of grease-free grade 0 steel wool (commercially available for scrubbing) was dipped in 50 ml freshly prepared acidified copper sulfate solution (mixture of 10% copper sulfate solution 5 ml, 10% Tween 80 w/v 5 ml, Sulfuric acid 2 mol/l 3 ml and distilled water up to 200 ml) until copper color appeared. Excess solution was drained and the steel wool was molded into a loose pad to fit an open Petri plate. A two inches long white-wax candle was placed at the center of this plate. A small test tube containing mixture of 0.5 g sodium-bicarbonate and 0.5 g magnesium carbonate was kept ready to put inside one 1000 ml test jar, just after placing inoculated plate. This 1000 ml jar holds three inoculated plates at a time while one 500 ml jar could be used for single plate incubation.
Stock culture isolates were grown on a Brucella blood agar (BBA) plate and subsequently sub-cultured in thioglycollate broth (Hi-Media, India). A loopful (0.01 ml) of the 24 hours thioglycollate broth culture was identically streaked on each prereduced culture plate used in the study.
The inoculated bi-layered Brucella blood agar plate (BL-BBA) was placed in the jar, keeping agar medium at the bottom. Then as rapidly as possible, the test tube containing CO 2 generator was placed at one side and the open plate containing treated steel wool and candle was placed on inoculated plate. About 2 ml of water was added quickly to the tube containing CO 2 generator and candle was lighted. Then jar lid was tightly closed [Figure 1]. Simultaneously in another two sets, same isolate was similarly streaked on normal BBA plate and one BL-BBA plate, and then both were placed in GasPak jar, keeping inoculated media on top in former and on bottom in the latter case. The Anaerogas Pack (HI-Media, India) was then activated before closing GasPak jar lid. The combustion jar and GasPak jar were simultaneously placed in 37°C incubator for 48 hours. Another set was simultaneously incubated aerobically for aero-tolerance study.
|Figure 1: Modified candle jar comprises of a transparent jar with air-tight lid, three bi-layered anaerobic media in Petri plates, an open plate containing acidifi ed steel wool and a white-wax candle, a test tube containing carbon-dioxide generator chemicals|
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Growth and colony size were was examined at 48 hours. The overall growth appearance and the average diameter of 10 well isolated, largest colonies were recorded by use of the following scale: 0 - no growth; 1 - haze; 2 - <0.5 mm; 3 - >0.5 mm; and 4 - >1 mm according to the scale used by Roe et al.,  The distribution of colony size scores on BBA in conventional GasPak system, on inverted BL-BBA in GasPak system and same in modified candle jar system was compared statistically by determining mean and standard deviation values using Graphpad Prism 5 version 5.00 statistical software. The number of colonies present in each plate was evaluated on a scale of 1+ to 4+, depending on presence of more than 10 colonies on original inoculating site and in first, second, and third streak areas.
| ~ Results|| |
The modified methylene blue indicator remained colorless till seventh day of experiment both in GasPak system and modified candle jar system. Test anaerobes failed to grow aerobically. After identical inoculations, the growth rate was seen to increase in GasPak system when bi-layered media was placed at the bottom instead of single layered medium at the top, and a still higher growth rate was noted when modified candle jar was used in place of GasPak system. The average size of isolated colonies was also larger in modified candle jar system than that of GasPak system [Table 1]. The reference strain anaerobes were maintained for 6 months by sub-culturing at 7 days intervals in modified candle jar system.
|Table 1: Distribution of colony size scores for anaerobes grown on conventional medium in GasPak, modifi ed medium in GasPak and the same in modifi ed candle jar system|
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| ~ Discussion|| |
Total avoidance of toxic oxygen exposure to anaerobes during collection of their clinical materials, transportation, inoculation, incubation, and handling is impossible. For this technical limitation only 1-5% of total bacterial population from mouse cecum could be isolated with anaerobic jar technique,  whereas 20-45% by cultivation in anaerobic glove boxes.  For different methods of anaerobiosis such unavoidable oxygen exposure time is different. Sometimes transit delay from collection to inoculation is longer particularly for a large scale processing unit like glove box or for a smaller unit like anaerobic jar that takes longer preparatory time. Bed side inoculation and placing single unit plate into a handy, sealed anaerobic chamber can minimize this delay. Although high isolation rate by any method of anaerobiosis depends upon the nutrient content and reduced state of medium but most importantly, on how early the process ensures attainment of a critical low level of oxygen to culture environment in a sustainable manner. Except for automated anaerobic glove box all other methods take much time to attain critical low level of oxygen in the chamber, which is several folds higher than the generation time of test anaerobes. This delay may be a major threat for viability of anaerobes present in the inoculums.
In the course of evolution of anaerobic methodology initial goals were removal or reduction of oxygen tension from confined environment for holding inoculated plates and maintenance of reduced state of the medium. Based on permutations and combinations of a few elementary principles, no fewer than 300 different kinds of anaerobic apparatus were described between 1888 and 1918.  Finally GasPak system was developed  as an acceptable method for most small-scale laboratories. However, it is costly and single use technique and takes about 90 minutes to attain a critical low level of oxygen, that is, around 0.2%,  which may be a factor for poor isolation rate.
As its alternative, candle jar technique can be applied for quick reduction of major bulk of oxygen and then residual 1-2% oxygen in the air can be slowly reduced by including a plate containing reducing chemicals. Such two steps reduction procedure may be a simpler and cheaper alternative for anaerobiosis. Several earlier workers applied various chemical methods for oxygen reduction with the goal of anaerobiosis including quick reduction of oxygen by ignited phosphorus or slow oxygen reduction by use of alkaline pyrogallol  or acidified copper coated steel wool.  Any single method is not good enough for anaerobiosis because rapid combustion usually ends with incomplete exhaustion of oxygen at the point of extinction of the flame, while slow combustion has its limitation for delayed response. Combination of rapid and slow combustion systems is expected to be more efficient.
By combustion of white-wax candle in candle jar system, 4-5% CO 2 is generated with instantaneous lowering of oxygen level inside sealed jar. The simple experiment for oxygen consumption by burning candle from water sealed inverted measuring cylinder showed 15-16% air volume replacement by water due to postcombustion vacuum created. But actual oxygen volume used up was at least 4% more due to added volume of CO 2 , which indicated 1-2% un-burnt oxygen from 21% oxygen normally present in the air.
In our two step combustion system acidified copper coated steel wool  is used for removal of residual oxygen after candle combustion. One gram iron wool absorbs approximately 1 ml of oxygen per minute. So from a 1000 ml sealed jar, postcombustion, 10-20 ml residual oxygen can be removed theoretically within 2-4 minutes if 5 g of treated steel wool is used. In our modified candle jar system we used this combination along with a CO 2 generator to elevate CO 2 level in the jar to approximately 10%. It also helped to replace stagnant air inside Petri-plates by heavier CO 2 so that their oxygen content could take part in slow combustion process. Although a similar technique was used in anaerobic bag culture method,  only steel wool was used there as combustion agent along with a CO 2 generator. That took 90 minutes to attain 0.7% oxygen level. Yet GasPak equivalent anaerobiosis was observed in anaerobic bag culture. As expected a more efficient anaerobiosis was noted in our two steps combustion procedure. Not only better growth of anaerobes with larger colonies was noted, but also a persistently low level of oxygen was indicated by methylene-blue indicator. For our technical limitation the time curve for oxygen level reduction could not be measured by mass-spectroscopic analysis of jar air samples. In our innovative system of anaerobiosis, further enhancement of growth was achieved by applying a technique for rapid dispersion of inhibitory metabolites accumulated around growing colonies. This was effected by incorporating a buffered-charcoal containing solid agar as the bottom layer of the bi-layered anaerobic medium. By adopting such simple measures anaerobe isolation was possible with minimal efforts similar to those required for isolation of most aerobes and also larger isolated colonies were obtained. Added advantage of the technique is that, desired anaerobiosis can be created even at collection point, also immediately after each handling, without much cost. The method is also suitable for antibiotic resistance tests for anaerobes. The same system can also be used for preparing prereduced medium by storing it for 48 hours in the candle jar system. The agents used for rapid and slow combustion can be appropriately substituted by other chemicals keeping in mind that reactions should not produce much inhibitory chemicals and oxygen utilization should be fast. Further evaluation is needed for testing suitability of the technique for isolation of different anaerobes from various clinical materials.
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