Three strains (labelled BC-Y2, bc-4 and BC-1) were isolated from the buccal tubes. Six buccal tubes were found to be contaminated with bacteria. No bacteria were isolated from saline without buccal tubes (data not shown). Based on phylogenetic analyses of 16S rRNA genes, the identities of these bacterial strains (BC-Y2, bc-4 and BC-1) are respectively: Micrococcus luteus, Staphylococcus haemolyticus and Acinetobacter calcoaceticus (Figure 1). The 16S rRNA gene sequences for BC-Y2, bc-4 and BC-1 were deposited in GenBank with the following access numbers: {“type”:”enter-nucleotides”,”attrs”:{“text”:”GU370962″,”term_id”:”284155443″,”term_text”:”GU370962″}}GU370962, {“type”:”enter-nucleotide”,”attrs”:{“text”:”GU300765″,”term_id”:”282205338″,”term_text”:”GU300765″}}GU300765 and {“type”:”enter-nucleotide”,”attrs”:{“text”:”GU370964″,”term_id”:”284155445″,”term_text”: “GU370964”}}GU370964, respectively. Micrococcus luteus has been shown to survive in oligotrophic environments for long periods of time. Recent work by Greenblatt et al. shows that, based on 16S rRNA analysis, Micrococcus luteus has survived for at least 34,000 to 170,000 years, possibly much longer. [2] It was sequenced in 2010 and has one of the smallest free Actinomycetota genomes sequenced to date, comprising a single circular chromosome of 2,501,097 bp. [3] Using standard methods, 10 g of each sample of sliced ham, chicken or minced meat was mixed with 90 ml of sterile peptone water solution (0.1%) for 1 min in a sterile gastric pouch (Stomacher Lab System Model 400; Colworth, London, UK) with a Stomacher (Colworth Stomacher 400; Colworth, London, United Kingdom). Subsequent dilutions were obtained by mixing 1 ml of aliquot homogenate with 9 ml of peptonic water solution (0.1%).
These dilutions were then analyzed in parallel by aerobic colony count on standard nutrient agar plates and counted visually after an incubation period of 48 h at 30 °C and by fluorescence-based dosing, where they were then delivered with the standard working oxygen sensor in microwaves and monitored as before on a plate reader to determine the times required to achieve threshold intensities. The CFU/ml of the agar plates were then compared to those calculated using calibration curves to convert threshold times to CFU/ml. In aerobic conditions, rapidly dividing cell cultures require more oxygen than slow-dividing cell cultures. To demonstrate the ability of the respirometric test to determine CFU in complex samples, it was applied to different types of food samples (homogenates) (n = 17) with unknown microbial numbers. All food samples from cooked ham, chicken and ground meat were successfully analyzed by respirometry at a measuring temperature of 30°C, allowing the growth of a variety of common aerobic bacteria. To avoid false-negative results (for example, due to reduced probe signals or accidental loss of device sensitivity), blanks with test samples without a probe were also included in the test. As expected, the spaces produced base signals significantly weaker than negative and positive, so the distinction between the latter two was reliable. The comparison of aerobic bacteria count results in food samples by respirometry (using three sets of calibrations) and by conventional method on agar plates is shown in Figure 4.4. We can see a good correspondence between the two methods. Of the three respirometric calibrations, the E.
coli One was statistically the least different from the data obtained with the conventional agar plate test. F-test analysis using OriginLab 7.5 software returned F-values of 0.07, 0.15 and 7.65 for calibration curves based on E. coli, M. luteus and P. fluorescens with corresponding P values of 0.9903, 0.9566 and 0.0003. The accuracy of CFU/ml calculated using the calibration curves of E. coli. coli and M. luteus show that foodborne bacteria have growth rates similar to these bacteria under the specified test conditions (30°C).
Oxygen tolerance is related to the bacteria`s ability to detoxify superoxide and hydrogen peroxide, which are produced as a byproduct of aerobic respiration. The growth of bacteria with different oxygen requirements in thioglycolate tubes is illustrated in Figure 9.29. In tube A, all growth can be seen at the top of the tube. Bacteria are obligate (strict) aerobes that cannot grow without an abundant supply of oxygen. Tube B looks the opposite of tube A. Bacteria grow at the bottom of the B tube. These are obligate anaerobes that are killed by oxygen. The C tube shows strong growth at the top of the tube and growth throughout the tube, a typical result in facultative anaerobes.
Facultative anaerobes are organisms that grow in the presence of oxygen, but also grow in the absence, relying on fermentation or anaerobic respiration when there is a suitable electron acceptor other than oxygen and the organism is capable of performing anaerobic respiration. The energy yield of anaerobic growth is lower than that of aerobic growth, so the cells below the top of the tube do not reach the same density. The aerotolerant anaerobes of the D tube are indifferent to the presence of oxygen. They do not use oxygen because they normally have a fermentative metabolism, but they are not damaged by the presence of oxygen, as are mandatory anaerobes. The growth pattern in the E tube is that of microaerophiles. They need a minimum level of oxygen for their growth, about 1% to 10%, well below the 21% in the atmosphere. Many mandatory anaerobes are found in the environment where anaerobic conditions prevail, such as deep bottom sediments, calm waters, and at the bottom of the deep ocean, where there is no photosynthetic life. Anaerobic conditions naturally also exist in the intestinal tract of animals. Obligate anaerobes, mainly bacterioids, account for a large proportion of microbes in the human gut. Transient anaerobic conditions are present when tissues are not supplied with blood; They die and become an ideal breeding ground for obligate anaerobes.