Influence of Lack of CO₂ on Anaerobic Bacteria: a Quick Method To Verify the Absence of CO₂

Abstract

Since CO₂ is carbonic acid gas, it will decrease the pH if added to water. Therefore, a simple test can determine whether CO₂ is present in a gas mixture. If the pH of gassed water does not change significantly, then CO₂ is not present in the gas.

Anaerobic organisms may fail to grow in anaerobic conditions for a variety of reasons. An incorrect temperature in the incubator, low humidity in the chamber, an inactive catalyst, an incorrect mixture of gas in the tank, the presence of toxic gas (e.g., O₂) in the chamber, or the lack of positive pressure are possibilities. When diminished growth of anaerobes is observed, a methodical investigation should be conducted to discover the problem(s). CO₂ is an essential component of the proper gas mixture for the growth of anaerobes. While improper gas mixture is a rare cause of growth failure, we present a fast, easy, low-tech method of verifying the absence of CO₂.

In the example described here, microbiologists observed that the recovery of anaerobes seemed to be decreased. Although Clostridium perfringens in clinical specimens continued to grow, the yield of the Bacteroides fragilis group in clinical specimens seemed lower than usual. However, this was not confirmed until the quality control (QC) organism B. fragilis ATCC 25282 failed to grow when a new shipment of media was tested. (At the same time, C. perfringens ATCC 13124 continued to grow for QC.) The yield of anaerobes by months is shown in Table 1. In July, a sharp drop occurred in the number of all anaerobes (including anaerobic gram-negative rods) isolated. (The percentage of positive anaerobe cultures dropped by approximately half.) Humidity, temperature, catalyst activity, pressure, and the presence of O₂ inside the anaerobic chamber were all investigated as possible causes of this growth failure. Since these parameters were within acceptable limits, a less likely cause, incorrect gas tank mixture, was investigated.

TABLE 1.

Percentage of positive anaerobic cultures

Group	% Positive during month of:
	Feb.	Mar.	Apr.	May	June	July	Aug.	Sept.	Oct.	Nov.	Dec.
All anaerobes	15.8	28.3	11.6	18.7	20.0	9.8	22.7	15.6	15.6	15.6	16.0
Anaerobic gram-negative bacilli	11.7	9.4	5.4	9.0	15.2	1.2	9.2	14.7	8.6	4.6	4.8

The composition of gas required for the Bactron anaerobic chamber is a trimix of nitrogen, hydrogen, and carbon dioxide (90, 5, and 5%, respectively).

CO₂ and water form carbonic acid gas as shown in the following equation:

Based on the fact that adding CO₂ to a nonbuffered solution such as water will decrease the pH, measuring the pH before and after adding gas to a solution will determine the absence of CO₂ in the gas. The procedure is as follows. (i) The pH of ∼50 ml of water in a 100-ml plastic beaker is measured by a pH meter and recorded. (ii) The distal tube connecting the gas in the tank to the chamber is disconnected and is crimped to stop the flow of gas. (iii) This distal end of the tube is inserted beneath the surface of the water in the beaker, and then the crimp is released so that gas flows into the water. (iv) After the gas has bubbled through the water for about 20 s, the pH of the gassed water is measured. If the gas contains CO₂, the pH of the water will drop significantly (e.g., from 8.0 to 4.2). A drop in pH from 8.0 to 4.2 represents nearly a 10,000-fold difference because pH units are on a log scale.

When the gas in the tank in use during July and early August (tank A) was tested, the pH dropped only from 8.0 to 7.2, signifying that there was little or no CO₂ in the gas tank. Subsequently, a new, previously unopened tank (tank B) with a different lot number was tested, and the pH dropped from 8.0 to 4.2, indicating the presence of CO₂. When this second tank was used to supply gas for the anaerobe chamber in late August, the QC organisms and anaerobes from clinical specimens grew as expected (Table 1).

After the gas mix was tested by the pH method, the gas in the tank was reanalyzed by the vendor. It was found to contain no CO₂. Tank A had been erroneously labeled as containing CO₂ and contained only a bimix of nitrogen and hydrogen.

While any gas with the potential to form an acid will cause a change in pH by this method, the effect of CO₂ on pH is a characteristic which can be useful in the clinical microbiology laboratory. Suppliers of gas generally offer two different types of contracts to clients. Either the gas mixture is analyzed and verified for each individual gas tank or the gas mixture isanalyzed for a batch of tanks, the latter procedure being more economical. The added cost of individual tank gas verification is ∼$50 per tank. Because of this cost difference, it is likely that most hospitals have a contract which accepts verification of batched gas tanks. The type of error described in this report is more likely to occur for clients who accept batched verification. Therefore, it is important that microbiologists know which type of agreement exists at their own institution.

In conclusion, the lack of CO₂ may inhibit the growth of anaerobes. Although incorrect gas mixture rarely occurs, a quick, in-house presumptive test based on a change in pH is available to determine the presence or absence of CO₂.