Method For Rapid Detection And Evaluation Of Cultured Cell Growth

Abstract

Provided is a method and system for the rapid and accurate detection of growth and metabolism of a cellular microorganism in a population of microorganisms in a non-liquid, culture medium. Further provided is a gelled culture medium containing a non-toxic, water-soluble, phosphorescent compound which measures oxygen content (partial pressure) of a microorganism also contained therein, by oxygen-dependent quenching of phosphorescence; or the gel contains a fluorescent pH indicator that demonstrates growth of the microorganism by pH-dependent intensity change or wavelength shift in the emission spectrum. Further provided is a system and method for killing undesirable microorganisms or colonies in the culture medium without harming the surrounding microorganisms.

1 . A method for rapidly detecting growth or metabolism of an immobilized anaerobic microorganism comprising:
 immobilizing a population of cells of at least one anaerobic microorganism in an aqueous gelling medium comprising a dissolved water soluble fluorescent pH indicator compound that is non-toxic to the microorganism;   thereby maintaining communicable contact between each cell and the fluorescent pH indicator in the surrounding medium, such that the quenchable fluorescent pH indicator is responsive to hydrogen ion changes in the medium;   exciting the dissolved fluorescent indicator to fluoresce, and   detecting a change in fluorescence emission by the fluorescent pH indicator in the gelled culture medium, wherein a change in fluorescence emission is indicative of a hydrogen ion change resulting from growth or metabolism of the microorganism.   
   
   
       2 . The method of  claim 1 , wherein the immobilizing step further comprises:
 mixing the aqueous gelling medium in sterile liquid form together with the population of cells of the at least one microorganism to form an inoculated mixture;   inserting the inoculated mixture into a space within a hollow form growth chamber; and   allowing the inoculated mixture to form a gel, thereby immobilizing the cells contained therein.   
   
   
       3 . The method of  claim 2 , further comprising selecting and removing from the gel at least one colony of the at least one microorganism detected by an increase in fluorescence indicative of growth or metabolism. 
   
   
       4 . The method of  claim 3 , further comprising propagating the selected and removed microorganism in a separate sterile culture medium. 
   
   
       5 . A hollow-form growth chamber for use in rapidly detecting growth or metabolism of a microorganism, comprising:
 a hollow form defining a space; and   an aqueous gelled culture medium within said space of said hollow form, wherein said gelled culture medium further comprises a population of cells of at least one microorganism and either a dissolved oxygen-quenchable phosphorescent compound or a dissolved fluorescent pH indicator compound.   
   
   
       6 . The chamber of  claim 5 , wherein said hollow form further comprises: two parallel plates defining a longitudinal plane of the space and at least two spacers disposed there between, thereby defining the space as rectangular. 
   
   
       7 . The chamber of  claim 5 , wherein the hollow form is preformed or premolded to define the requisite space. 
   
   
       8 . The chamber of  claim 5 , wherein the chamber comprises a sterile, disposable, puncture-resistant plastic culture bag. 
   
   
       9 . The chamber of  claim 5 , wherein the hollow form further comprises a sterile, disposable, puncture-resistant plastic culture bag inserted therein 
   
   
       10 . A system for killing one or more undesirable microorganism(s) immobilized in a gel within a growth chamber, wherein the system comprises:
 an excitation light source,   a light detector,   a collimated laser diode,   a means for positioning a growth chamber device relative to said collimated laser diode, and   a processing means operably interconnecting the excitation light source, the light detector and the collimated laser diode.   
   
   
       11 . The system of  claim 10 , wherein the excitation light source excites a photoluminescent molecule within the growth chamber, the light detector detects light emitted from the photoluminescent molecule, and the processing means aims the collimated laser diode in response to the detected light emission and activates the laser diode to an intensity sufficient to kill one or more microorganism(s) immobilized within the growth chamber. 
   
   
       12 . A method of killing one or more undesirable microorganism(s) immobilized within the growth chamber of system of  claim 10  without harming other desirable microorganisms contained therein, wherein the method comprises:
 activating the excitation light source and exciting a photoluminescent molecule to luminescence within the growth chamber;   detecting with the light detector, emitted light from the photoluminescent molecule;   aiming the collimated laser diode by the processing means in response to the detected emitted light;   activating the laser diode to an intensity sufficient to kill the one or more undesirable microorganisms immobilized within the growth chamber; and then   killing the one or more undesirable microorganisms, without harming desirable microorganisms immobilized within the growth chamber, thereby permitting continued growth and metabolism of the unharmed microorganisms.   
   
   
       13 . The method of  claim 12 , wherein the microorganism(s) to be killed is an undesired microorganism within a population of selected microorganisms, said method further comprises, prior to activating the excitation light:
 mixing an aqueous gelling medium in sterile liquid form together with a population of microorganisms to form an inoculated mixture, which may contain one or more undesirable microorganism(s);   inserting the inoculated mixture into a space within the hollow form growth chamber;   allowing the inoculated mixture to gel, thereby immobilizing the cells contained therein; then after the gel has formed,   determining if the population of cells comprises one or more undesirable microorganisms.   
   
   
       14 . A kit for use in rapidly detecting growth or metabolism of at least one immobilized microorganism comprising:
 an aqueous gelling medium in powdered form that gels after solublizing;   a non-toxic, aqueously soluble, oxygen-quenchable phosphorescent compound or fluorescent pH indicator, or both;   a growth chamber; and   instructional material.   
   
   
       15 . The method of  claim 4 , wherein the separate culture medium further comprises an antibiotic. 
   
   
       16 . The method of  claim 1 , wherein the microorganism is selected from the group consisting of species from the genera  Bacillus, Mycobacterium, Actinomyces, Nocardia, Pseudomanas, Methanomonas, Protaminobacter, Methylococcus, Arthrobacter, Methylomonas, Brevibacterium, Acetobacter, Micrococcus, Rhodopseudomonas, Corynebacterium, Microbacterium, Achromobacter, Methylobacterium, Methylosinum, Methylocystis, Acinetobacter, Escherichia , and mixtures thereof. 
   
   
       17 . The method of  claim 16 , wherein the microorganism is further selected from the group consisting of the tuberculosis agents  Mycobacterium tuberculosis, M. boris  and  M. avium