Screening for l-form bacteria

Abstract

Disclosed herein are methods for screening clinical or biological samples to determine the presence of L-form bacteria within the sample. Methods include contacting a sample to a liquid growth medium and incubating the liquid growth medium at a temperature lower than 37 degrees C. The liquid growth medium is monitored for L-form bacterial growth. An amount of the liquid growth medium is transferred as an inoculant to a solid growth medium, and the solid growth medium is incubated under conditions that maintain a hydrated state of the inoculant to enable the L-form bacteria to efficiently interface with the solid growth medium and continue to grow.

1 . A method of screening a sample to determine the presence of L-form bacteria within the sample, the method comprising:
 contacting a sample to a first growth medium;   incubating the first growth medium under a first set of incubation conditions;   transferring at least a portion of the first growth medium, as an inoculant, to a second growth medium under conditions that maintain a hydrated state of the inoculant;   incubating the second growth medium under a second set of incubation conditions; and   monitoring the second growth medium for the presence of bacteria.   
     
     
         2 . The method of  claim 1 , further comprising isolating at least one bacterial strain grown on the second growth medium and testing the bacterial strain against one or more antimicrobial compounds. 
     
     
         3 . The method of  claim 1 , further comprising isolating at least one bacterial strain grown on the second growth medium and using the bacterial strain for the production of an antibody, a vaccine, or a diagnostic reagent. 
     
     
         4 . The method of  claim 1 , wherein the first growth medium is a liquid, and wherein the second growth medium is a solid. 
     
     
         5 . The method of  claim 4 , wherein the inoculant is added to a surface of the second growth medium, the method further comprising placing an insert over the inoculant to seal at least a portion of the inoculant between the surface of the second growth medium and the insert. 
     
     
         6 . The method of  claim 5 , wherein the second set of incubation conditions includes a first solid-phase incubation time period and a second solid-phase incubation time period, the second growth medium being positioned inoculant side down for the first solid-phase incubation time period and inoculant side up for the second solid-phase incubation time period. 
     
     
         7 . The method of  claim 1 , wherein the first and second growth media are individually selected from the group consisting of: mannitol salt, Kligler iron, Vogel Johnson, Columbia blood, brain heart infusion (BHI), nutrient, bovine serum, and human serum. 
     
     
         8 . The method of  claim 1 , wherein at least one of the first and second growth media is a complex media. 
     
     
         9 . The method of  claim 1 , wherein at least the first growth medium omits polymerase chain reaction (PCR) inhibiting substances and substances inhibiting the reversion of L-form bacteria to classic form. 
     
     
         10 . The method of  claim 1 , wherein the first set of incubation conditions includes an incubation temperature lower than 37 degrees C. 
     
     
         11 . The method of  claim 1 , further comprising comminuting the sample prior to transferring the sample to the second growth medium in order to increase culture growth and/or decrease culture time. 
     
     
         12 . The method of  claim 11 , wherein comminution is performed in a comminuting container containing a comminuting media, the comminuting media being configured to contact portions of the sample to increase exposure of L-form bacteria within the sample to surrounding growth media. 
     
     
         13 . The method of  claim 1 , wherein the first growth medium is incubated in an atmosphere having a relative humidity maintained above 40%. 
     
     
         14 . A method of screening a sample to determine the presence of L-form bacteria within the sample, the method comprising:
 contacting a sample to a first growth medium within a comminuting container;   comminuting the sample;   incubating the sample under a first set of incubation conditions; and   monitoring the first growth medium for the presence of L-form bacteria.   
     
     
         15 . The method of  claim 14 , further comprising transferring at least a portion of the comminuted first growth medium, as an inoculant, to a second growth medium, incubating the second growth medium under a second set of incubation conditions, and monitoring the second growth medium for the presence of bacteria. 
     
     
         16 . The method of  claim 14 , wherein the first growth medium is a liquid, and wherein the second growth medium is a solid. 
     
     
         17 . The method of  claim 14 , wherein the inoculant is added to a surface of the second growth medium, the method further comprising placing an insert over the inoculant to seal at least a portion of the inoculant between the surface of the second growth medium and the insert. 
     
     
         18 . The method of  claim 14 , wherein the first set of incubation conditions includes an incubation temperature lower than 37 degrees C. 
     
     
         19 . The method of  claim 14 , wherein the first growth medium is incubated in an atmosphere having a relative humidity maintained above 40%. 
     
     
         20 . A sample collection device for collecting samples to be tested for the presence of L-form bacteria, the sample collection device comprising:
 a sample carrier configured to receive and associate with a sample;   a passage including at least one surface configured to extract a portion of the sample from the sample carrier as the sample carrier is contacted with the passage;   a first growth area configured to receive a portion of the sample extracted from the sample carrier, the first growth area being configured to culture bacteria within the sample; and   a second growth area configured to receive a portion of the sample carrier after the sample carrier has passed through the passage, the second growth area configured to culture bacteria within the sample.