Bacterial population detection is based on detection of adenosine triphosphate (ATP), chemical present in all living matter. Amount of ATP in sample, after chemically removing all nonbacterial ATP, is directly related to bacterial population. Sensitivity is improved by concentration step; specificity is improved by lowering pH of solution

Picciolo, G. L.

English

NASA Technical Reports Server

*	 December 1974	 B74-10231 
NASA TECH BRIEF 
•	 Goddard Space Flight Center 
NASA Tech Briefs announce new technology derived from the U.S. space program. They are issued to encourage 
commercial application. Tech Briefs are available on a subscription basis from the National Technical Information 
Service, Springfield, Virginia 22151. Requests for individual copies or questions relating to the Tech Brief program may 
be directed to the Technology Utilization Office, NASA, Code KT, Washington, D.C. 20546. 
Improved Methods for Counting Bacteria in Physiological Fluids 
fl
The problem: 
Most methods used for counting bacteria in physio-
logical fluids (e.g., blood and urine) rely on the growth 
of the bacteria and subsequent visual counting of 
colonies. Normally, a fluid sample is mixed with a 
nutrient medium and allowed to grow. Culture growth 
may take up to several days, delaying treatment. 
The solution: 
A method for detecting bacterial population that 
does not depend on growth is based on the detection 
of adenosine triphosphate (ATP), a chemical present in 
all living matter. The amount of ATP in a sample, after 
chemically removing all nonbacterial ATP, is directly 
related to the bacterial population. This method im-
proves on the method described in the NASA Tech Brief 
B71-10051 (GSC-11092) in two ways. The sensitivity 
is improved by a concentration step, thereby detecting 
the presence of lower levels of bacteria. The specificity 
is improved by lowering the pH of the solution to 4.0, 
eliminating the measurement of nonbacterial ATP. 
Bacterial ATP can be measured even in the presence of 
as much as 106 leucocytes per ml. 
How it's done: 
Infected fluids, for example urine, include non-
bacterial cells, such as epithelial cells, erythrocytes, 
leucocytes, and renal cells. All of these contain ATP, 
as does bacteria. Soluble ATP also is excreted in the 
urine. Therefore, an important step in the bacterial ATP 
detection process is to first destroy the ATP associated 
with the nonbacterial cells and soluble ATP and leave 
only bacterial ATP.
The nonbacterial cells are ruptured by a nonionic 
detergent such as octyl phenoxy polyethoxyethanol 
which does not affect bacterial cells. This process 
requires approximately ten minutes. The bacteria are 
concentrated by centrifugation. Next, the soluble ATP is 
destroyed by adding apyrase, an enzyme that hydrolyzes 
the freed and soluble ATP. The pH of the solution is 
lowered by the addition of malate buffer pH 3.75. 
This desorbs the ATP bound to particulates and allows 
the apyrase to hydrolyze the freed ATP, thus allowing a 
more accurate determination of the bacterial ATP which 
will subsequently be freed by acid extractant. The 
apyrase then is destroyed by adding a strong inorganic 
acid (e.g., nitric acid). When the acid is added, the 
bacterial cells are ruptured, freeing their ATP. The 
acid extractant then is diluted with distilled water. 
At this point, the only remaining ATP in the 
suspension is bacterial. A firefly luciferase-luciferin. 
mixture in soluble form and a small quantity of 
magnesium sulfate are added to the suspension. The 
luciferase is an enzyme which catalyzes the reaction of 
luciferin with ATP in the presence of magnesium ions. 
The reaction of the luciferin with the bacterial ATP 
produces light, the intensity of which is directly 
proportional to the amount of bacteria present. Light 
intensity is measured directly using a photometer. 
Amounts of chemically synthesized ATP are used for 
calibration. This procedure does not rely on growth and 
can be accomplished in less than one hour. It can detect 
the presence of as few as 1000 bacteria/ml from 10 ml 
of sample.
(continued overleaf) 
This document was prepared under the sponsorship of the National 	 Government assumes any liability resulting from the use of the 
Aeronautics and Space Administration. Neither the United States 	 information contained in this document, or warrants that such use 
Government nor any person acting on behalf of the United Slates 	 will be free from privately owned rights.
https://ntrs.nasa.gov/search.jsp?R=19740000231 2020-03-17T13:55:02+00:00Z
Note: 
Requests for further information may be directed to: 
Technology Utilization Officer 
Goddard Space Flight Center 
Code 207.1 
Greenbelt, Maryland 20771 
Reference: TSP74-1023 1
Patent status: 
This invention is owned by NASA and a patent 
application has been filed. Inquiries concerning non-
exclusive or exclusive license for its commercial develop-
ment should be addressed to: 
Patent Counsel 
Goddard Space Flight Center 
Code 204 
Greenbelt, Maryland 20771 
Source: Grace L. Picciolo and 
Emmett W. Chappelle
Goddard Space Flight Center 
(GSC. 1 1917)
is 
B74-10231	 Category 05,03


Improved methods for counting bacteria in physiological fluids

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