Detection of catalase-containing bacteria

• US 4,065,357 A
• Filed: 11/17/1975
• Issued: 12/27/1977
• Est. Priority Date: 01/21/1974
• Status: Expired due to Term

• Current Assignee: General Electric Company
• Original Assignee: General Electric Company
• Inventors: Groves, James N.
• Primary Examiner(s): Tanenholtz, Alvin E.
• Assistant Examiner(s): Kepplinger, Esther M.
• Application Number:
  US05/632,483
• Time in Patent Office: 771 Days
• Field of Search: 195/103.5 R, 204/195 P
• US Class Current: 435/27
• CPC Class Codes: C12M 41/34 of gas
• CPC Class Codes: C12M 41/36 of biomass, e.g. colony counters or by turbidity measurements electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404
• CPC Class Codes: C12M 41/40 of pressure
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/30 involving catalase

Abstract

Sensitive, fast-response apparatus is described for the detection of the presence of (a) aerobic or facultatively anaerobic bacteria, (b) certain body cell breakdown products in body fluids or (c) unusual contamination of the ambient by aerobic bacteria. The method employed, which enables dynamic, continuous observation of catalase-H₂ O₂ reaction, is based upon measuring the incremental increase of oxygen partial pressure within a hydrogen peroxide solution. The inherent catalase content in such bacteria and in most animal cells promotes the rapid decomposition of hydrogen peroxide resulting in the liberation of oxygen. This measurement of oxygen partial pressure is made directly from the hydrogen peroxide solution by means of an oxygen permeable membrane polarographic cell equipped with a cathode configuration providing improved sensitivity. The presence of small numbers of bacteria or other catalase-containing cells may be quantitatively determined by conducting the catalase-H₂ O₂ reaction in a very small reproducible reaction volume.

Claims

1. A process for the clinical detection of the catalase activity in catalasa-containing cells in body fluid by dynamic monitoring of oxygen gas produced by the catalase-peroxide reaction comprising the steps of:

a. secruing a quantity of body fluid of unknown catalase content,

b. intorducing a predetermined volume of said body fluid into a non-expandable reaction volume separated by an oxygen permeable membrane from electrochemical measuring means, said electrochemical measuring means being in association with said oxygen permeable membrane said means being totally outside said closed reaction volume, said membrane being impermeable to hydrogen peroxide and having an oxygen permeability coefficient of at least about 5 ×

10^-11 gms./atmos.-cm.-sec.,

c. introducing a predetermined quantity of hydrogen peroxide solution into said reaction volume such that the volumes of said body fluid and hydrogen peroxide solution completely fill said reaction volume and such that the resulting solution has a hydrogen peroxide concentration in the range of from 0.01 to 1 mole per liter of said resulting solution, closing said reaction volume and

d. electrochemically measuring the increase of partial pressure of oxygen in said reaction volume by measuring the extent of diffusion of oxygen through said membrane, said extent of diffusion of oxygen through said membrane being linearly proportional to said partial pressure of oxygen in said reaction volume.

2. The process for the clinical detection of the presence of catalase-containing cells in said body fluid as recited in claim 1 wherein the volume of hydrogen peroxide solution introduced does not exceed about 10% of the volume of said body fluid.

3. The process for performing rapid, quantitative determination of catalase content by dynamic monitoring of the oxygen gas produced by the catalase-peroxide reaction comprising the steps of:

a. isolating two substantially identical separate portions of material of unknown catalase content in solution to be tested for catalase activity,

b. introducing a predetermined volume of each of said portions of said material into first and second reaction volumes, each of said reaction volumes being non-expandable and each being separated by an oxygen permeable membrane from a polarographic cell connected in series opposition, each said membrane being adjacent to the cathode of said cell and being impermeable to hydrogen peroxide and having an oxygen permeability coefficient of at least about 5 ×

10^-11 gms./atmos.-cm.-sec.,

c. inactivating the catalase content of one of said portions of said material without affecting the activity of other catalytic agents for hydrogen peroxide decomposition,

d. introducing a predetermined volume of identical hydrogen peroxide solution into both said reaction volumes such that the volumes of said material and hydrogen peroxide completely fill each of said reaction volumes and such that the resulting solution in each said reaction volume has a hydrogen peroxide concentration in the range of from 0.01 to 1 mole per liter of said resulting solution, closing said reaction volumes, said polarographic cell being totally outside said closed reaction volumes, and

e. measuring the difference between the increase of partial pressure of oxygen in said reaction volumes by means of said polarographic cell by measuring the extent of diffusion of oxygen through each said membrane, said extent of diffusion of oxygen through each said membrane being linearly proportional to the corresponding said partial pressure of oxygen in said reaction volumes.

4. The process for performing rapid, quantitative determinations of catalase activity as recited in claim 3 wherein the volume of hydrogen peroxide solution added does not exceed about 10% of the volume used of each of said portions of material.