Patent Grant
10379016
The disclosure of the present patent application relates to microbial culturing, and particularly to an apparatus for inoculating agar plates with microorganisms.
In microbiology, agar plates are used for several purposes, for example: disease diagnosis, susceptibility testing, estimating concentration of microorganisms in a sample, to isolate pure colonies, screening experiments, microbial detection and identification. To prepare an agar plate, the molten agar medium is poured into an agar plate and solidified by cooling. A microbial suspension is applied on the surface of agar media using different techniques. Some examples of these techniques include streak plating, spread plating, pour plating, and soft agar overlays. In streak and spread plating techniques, the microbial inoculum is transferred onto the agar plate and then spread over the surface of the media manually using an inoculator (a wire loop or glass loop or disposable plastic loop, for streaking) or a spreader (an L-shaped glass or metal rod, or cotton swab, or pre-sterilized glass beads, for spreading the microbes). While spreading or streaking the microbial inoculum on agar surface, problems with uneven spreading and discontinuous streaking of the microbes may lead to failed experiments and waste of time, as well as material. Another problem caused by wire loop, glass spreader, or cotton swab application is damaging the surface of the solidified agar media, which may occur when manually streaking or spreading the culture media. Sometimes, it is also observed that the culture did not reach the inner peripheral areas of the agar plate when using the L-shaped spreader. If microbial inoculum is absorbed in the center of the plate, it leads to condensed colonies in the center of the plate, which cannot be distinguished properly. The manual inoculation of agar plates by spreading or streaking technique is a slow process and requires a lot of attention and care to avoid damage to the agar surface. Accordingly, both manual spreading and streaking are time-consuming techniques.
Thus, an apparatus for inoculating agar plates solving the aforementioned problems is desired.
The apparatus for inoculating agar plates includes a spray chamber having an upper opening for receiving an atomized microbial suspension and a lower opening for receiving an agar plate. The apparatus also includes an atomizer including a reservoir and a fluid tube for delivering the microbial suspension to the atomizer nozzle. A containment feature extends around an inner surface of the spray chamber to catch any drops that may form on its inner wall and advance down towards the lower opening. The spray chamber allows multiple agar plates to be quickly inoculated without cross-contamination of agar habitats, without contaminating the outside of the plates, and without contaminating the work area.
The apparatus is used by positioning an agar plate within the lower opening of the spray chamber. The atomizer is secured to the top of the spray chamber so that an atomized microbial suspension may be sprayed through the upper opening of the spray chamber and onto the agar. Any drops that form on the inner surface of the spray chamber are caught by the containment feature.
These and other features of the present disclosure will become readily apparent upon further review of the following specification and drawings.
Similar reference characters denote corresponding features consistently throughout the attached drawings.
The apparatus for inoculating agar plates includes a spray chamber having an upper opening for receiving an atomized microbial suspension and a lower opening for receiving an agar plate. The apparatus also includes an atomizer including a reservoir and a fluid tube for delivering the microbial suspension to the atomizer nozzle. A containment feature extends around an inner surface of the spray chamber to catch any drops that may form on its inner wall and advance down towards the lower opening. The spray chamber allows multiple agar plates to be quickly inoculated without cross-contamination of agar habitats, without contaminating the outside of the plates, and without contaminating the work area.
A first embodiment of an apparatus and method for inoculating agar plates is shown in
An annular “J” shaped inner projection 102 or lip is located on the inner surface of spray chamber 101 close to lower opening 104. The projection 102 extends around the entire periphery of the inner wall resulting in a seamless barrier. The level of inner projection 102 on the inner surface of spray chamber 101 is such that the inner projection 102 is just above the vertical wall 121 of agar plate 120 (a petri dish with agar media contained therein), or just touches the top of the vertical wall 121 of the agar plate 120. Close proximity between the top of the agar plate wall 121 and the projection 102 does not allow atomized microbes to reach the wall of the spray chamber 101 below the projection 102. The projection 102 is slightly obliquely upward, as presented in
The inner projection 102 may include a layer of absorbent or adsorbent material 103 on its upper surface. While the microbial suspension is sprayed over the agar surface 122, some of the spray droplets may fall on the inner wall of the spray chamber 101 above the projection 102. When a large number of agar plates are inoculated, the small droplets on the inner wall of the spray chamber 101 will form large drops 142. After attaining the sufficient size or weight, the drops 142 will descend or roll down the inner wall of the spray chamber 101 towards the agar plate 120, as depicted in
While the apparatus 100 is not in use, the lower opening 104 and upper opening 106 of the spray chamber 101 may be sealed with the covers or closure elements 105 and 107, respectively.
Any agar media known in the art for culturing microorganisms is contemplated with the apparatus, while Mueller-Hinton Agar is preferred. Other types of agar may include Tryptic Soy Agar, Chocolate Agar, Thayer-Martin Agar, MacConey Agar, Eosin-methylene Blue Agar, Hektoen Agar, Mannitol Salt agar, and Sheep Blood Agar. Specific agars can be selected based on the type of bacteria being cultivated and the purpose of the cultivation.
The microbial suspension for inoculation is filled in the reservoir 116 of the atomizer 110, shown in
The atomizer 110 includes an atomizer nozzle, and through the application of pressure and gas, transforms the liquid stream into many small droplets. Preferably, the atomizer 110 is designed to produce a conical spray stream that creates a circular spray pattern. A fluid tube 115 is attached to the inlet of the atomizer 111 for drawing liquid from the bottom of the reservoir 116. It is contemplated that the fluid tube 115 be curved towards the direction of the bottom of the reservoir 116 so it can draw maximum fluid, indicated by dash lines 150 in
The apparatus for inoculating agar plates quickly and uniformly inoculates the microorganism on the surface of agar plate. Almost the entire surface of the agar is inoculated. The apparatus was tested for microbe growth on an agar plate after a single actuation of the atomizer and after double actuation of the atomizer. Double actuation produces more dense and uniform microbial growth. However, the microbial growth from a single actuation may be suitable in many cases. Additionally, inoculation was completed successfully without causing any damage to the surface of solidified agar media.
The number of colonies grown on the surface of agar plate will depend on the density of inoculum (number of CFU/ml) and the numbers of actuations applied, and the volume delivered by the actuator. If the density of inoculum (number of CFU/ml) is low (1-2×103 to 1-2×105 CFU/mL), then a higher number of actuations is required to provide uniformly dense growth over the agar plate, but if the density of inoculum is higher (1-2×105 CFU/mL), then single or double actuation may be sufficient to achieve the required dense and uniform growth on the surface of the plate.
The density and uniformity of microbial growth on the plate surface will be influenced by: number of colony forming units in the inoculum, number of atomization sprays, volume of inoculum delivered by the atomizer per atomization, the atomization efficiency of the atomizer, the distance between the nozzle of the atomizer and the agar surface, and angle of atomization. One can optimize the number of actuations required based on these parameters.
A method for inoculating an agar plate includes positioning an agar plate 120 within the lower opening of a spray chamber 101, having a ridge 102, and an absorbing material 103, as seen in
In an experiment, Mueller-Hinton Agar plates were prepared. Forty grams of Mueller-Hinton Agar (MHA) was suspended in 1 liter distilled water and then boiled to dissolve the powder completely. The medium was sterilized by autoclaving at 121° C. for 15 min. After autoclaving, the medium was cooled to 45° C. and poured 25 mL molten agar into sterile 90 mm diameter Petri dishes (petri plates) to give a depth of about 4 mm. The surface of the agar was dried to remove excess moisture before use.
The inoculum (microbial suspension) of S. aureus strain ATCC 2913 was prepared by selecting three to five morphologically similar colonies from overnight growth (16-24 h of incubation) on blood agar medium with a sterile loop and suspending the colonies in sterile normal saline to the density of a McFarland 0.5 standard, approximately equivalent to 1-2×108 CFU/mL. The suspension density was measured by using an UV spectrophotometer. The density of the suspension was adjusted to McFarland 0.5 by addition of saline or more organisms. The number of colony forming units (CFU/ml) in the culture samples plays an important role in the incubation of the agar plates. For colony isolation, diluted microbial suspension (about 1-2×103 CFU/ml) can be sprayed over agar plate.
For comparison, some agar plates were inoculated by cotton swab. A sterile cotton swab was dipped into the bacterial suspension (1-2×108 CFU/ml) and the excess fluid removed by turning the swab against the inside of the tube to avoid over-inoculation of plates. The inoculum was spread evenly over the entire surface of the agar plate by swabbing in three directions.
Other agar plates were inoculated by using the present apparatus. The agar plates were inoculated with inoculum of bacterial suspension (1-2×108 CFU/ml). The bacterial suspension was filled in the reservoir of the present apparatus and then sprayed uniformly using the atomizer over an agar plate held inside the spray chamber using the atomizer.
Antimicrobial susceptibility tests of the S. aureus strain ATCC 29213 was determined by the disc diffusion method according to the recommendations and guidelines of Clinical Laboratory Standard Institutes (CLSI M100-S24; 2014). A panel of commercial antibiotic discs comprising Gentamicin (GM), Amikacin (AN), Chloramphenicol (C), Imipenem (IPM), Tetracycline (TE), Cefepime (FEP), Sulfamethoxazole/trimethoprim (SXT), Aztreonam (ATM) was used for susceptibility testing. All commercial antibiotic discs were purchased from BBL (Becton Dickinson, USA).
The predetermined panel of antimicrobial disks was applied firmly on the surface of inoculated Mueller-Hinton agar media by using a disc dispenser device.
Within 15 min of application of antimicrobial disks, the plates were inverted and incubated aerobically at 35-37° C. for 18-20 h.
After incubation, inhibition zones were read at the point where no obvious growth is detected by the unaided eye when the plate was held about 30 cm from the eye. The inhibition zone diameters were measured to the nearest millimeter with a ruler. The plates were read from the back of the plate with reflected light against a dark background. Zone diameters of antibiotics in swab inoculated plate were compared with zone diameter in apparatus-inoculated plates. The results of zone diameters are presented in table 1.
As indicated by Table 1, the spray inoculation created similar results to the swabbing technique. The differences in results are minor, especially when comparing the double-actuated results. Differences of 1-2 mm are normal for separate zones of inhibition and are not indicative of substantial differences in antimicrobial activities. Therefore, when properly optimized, the spray technique discussed above will produce equivalent results to currently known techniques, such as swabbing, in a fraction of the time.
It is to be understood that the apparatus for inoculating agar plates is not limited to the specific embodiments described above, but encompasses any and all embodiments within the scope of the generic language of the following claims enabled by the embodiments described herein, or otherwise shown in the drawings or described above in terms sufficient to enable one of ordinary skill in the art to make and use the claimed subject matter.
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| 107022476 | Aug 2017 | CN |
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| 101389628 | May 2014 | KR |
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