Various methods of streaking
Pour Plate Method
This method involves plating of diluted samples mixed with melted agar medium. The main principle is to dilute the inoculum in successive tubes containing liquefied agar medium so as to permit a thorough distribution of bacterial cells within the medium. Here, the mixed culture of bacteria is diluted directly in tubes containing melted agar medium maintained in the liquid state at a temperature of 42-45°C (agar solidifies below 42°C).
The bacteria and the melted medium are mixed well. The contents of each tube are poured into separate Petri plates, allowed to solidify, and then incubated. When bacterial colonies develop, one finds that isolated colonies develop both within the agar medium (subsurface colonies) and on the medium (surface colonies). These isolated colonies are then picked up by inoculation loop and streaked onto another Petri plate to insure purity.
Pour plate method has certain disadvantages as follows: (i) the picking up of subsurface colonies needs digging them out of the agar medium thus interfering with other colonies, and (ii the microbes being isolated must be able to withstand temporary exposure to the 42-45° temperature of the liquid agar medium; therefore this technique proves unsuitable for the isolation of psychrophilic microorganisms.
However, the pour plate method, in addition to its use in isolating pure cultures, is also used for determining the number of viable bacterial cells present in a culture.
Figure 6. Pour plate method
Spread Plate Method
In this method the mixed culture of microorganisms is not diluted in the melted agar medium (unlike the pour plate method); it is rather diluted in a series of tubes containing sterile liquid, usually, water or physiological saline. A drop of so diluted liquid from each tube is placed on the centre of an agar plate and spread evenly over the surface by means of a sterilized bent-glass-rod.
The medium is now incubated. When the colonies develop on the agar medium plates, it is found that there are some plates in which well-isolated colonies grow. This happens as a result of separation of individual microorganisms by spreading over the drop of diluted liquid on the medium of the plate.
Figure 7. Spread plate method
Control questions:
1. Isolation of accumulative culture from pure cultures?
2. Principles of spread plate method?
3. Principles of pour plate method?
4. Principles of streak plate method?
Laboratory work № 7 Control for cultivation. Antimicrobial factors.
Purpose of work:
Studying of characteristics of the microorganisms which are carrying out biosynthesis of antibiotics (on an example of lysozyme), and sensitivity of microorganisms to antibiotics. Definition of a spectrum of antimicrobic action of antibiotics.
Materials and equipment:
Petri dishes with dried MPA medium, the sterile wadded tampon moistened with suspension investigated culture, sterile tweezers on an agar, display paper disks (4-5 pieces).
Antibiotic resistance is a type of drug resistance where a microorganism is able to survive exposure to an antibiotic. While a spontaneous or induced genetic mutation in bacteria may confer resistance to antimicrobial drugs, genes that confer resistance can be transferred between bacteria in a horizontal fashion by conjugation, transduction, or transformation. Thus, a gene for antibiotic resistance that evolves via natural selection may be shared. Evolutionary stress such as exposure to antibiotics then selects for the antibiotic resistant trait. Many antibiotic resistance genes reside on plasmids, facilitating their transfer. If a bacterium carries several resistance genes, it is called multidrug resistant (MDR) or, informally, a superbug or super bacterium.
Genes for resistance to antibiotics, like the antibiotics themselves, are ancient. However, the increasing prevalence of antibiotic-resistant bacterial infections seen in clinical practice stems from antibiotic use both within human medicine and veterinary medicine. Any use of antibiotics can increase selective pressure in a population of bacteria to allow the resistant bacteria to thrive and the susceptible bacteria to die off. As resistance towards antibiotics becomes more common, a greater need for alternative treatments arises. However, despite a push for new antibiotic therapies there has been a continued decline in the number of newly approved drugs. Antibiotic resistance therefore poses a significant problem.
Although there were low levels of preexisting antibiotic-resistant bacteria before the widespread use of antibiotics, evolutionary pressure from their use has played a role in the development of muiltidrug resistance varieties and the spread of resistance between bacterial species. In some countries, antibiotics are sold over the counter without a prescription, which also leads to the creation of resistant strains. In medicine, the major problem of the emergence of resistant bacteria is due to misuse and overuse of antibiotics. Other practices contributing towards resistance include the addition of antibiotics to livestock feed. Household use of antibacterials in soaps and other products, although not clearly contributing to resistance, is also discouraged (as not being effective at infection control). Also unsound practices in the pharmaceutical manufacturing industry can contribute towards the likelihood of creating antibiotic-resistant strains. The procedures and clinical practice during the period of drug treatment are frequently flawed - usually no steps are taken to isolate the patient to prevent re-infection or infection by a new pathogen, negating the goal of complete destruction by the end of the course.
Certain antibiotic classes are highly associated with colonisation with "superbugs" (highly antibiotic resistant bacteria) compared to other antibiotic classes. The risk for colonisation increases if there is a lack of sensitivity (resistance) of the superbugs to the antibiotic used and high tissue penetration, as well as broad-spectrum activity against "good bacteria". In the case of MRSA, increased rates of MRSA infections are seen with glycopeptides, cephalosporins and especially quinolones. In the case of colonisation with Clostridium difficile the high risk antibiotics include cephalosporins and in particular quinolones and clindamycin.
There is evidence that naturally occurring antibiotic resistance is common. The genes that confer this resistance are known as the environmental resistome. These genes may be transferred from non-disease-causing bacteria to those that do cause disease, leading to clinically significant antibiotic resistance.
In 1952 an experiment conducted by Joshua and Esther Lederberg showed that penicillin-resistant bacteria existed before penicillin treatment. While experimenting at the University of Wisconsin-Madison, Joshua Lederberg and his graduate student Norton Zinder also demonstrated preexistent bacterial resistance to streptomycin. In 1962, the presence of penicillinase was detected in dormant Bacillus licheniformis endospores, revived from dried soil on the roots of plants, preserved since 1689 in the British Museum. Six strains of Clostridium, found in the bowels of William Braine and John Hartnell (members of Franklin Expedition) showed resistance to cefoxitin and clindamycin. It was suggested that penicillinase may have emerged as a defense mechanism for the bacteria in their habitats, as in the case of penicillinase-rich Staphylococcus aureus, living with penicillin-producing Trichophyton. This, however, was deemed circumstantial. Search for a penicillinase ancestor has focused on the class of proteins that must be a priori capable of specific combination with penicillin. The resistance to cefoxitin and clindamycin in turn was speculatively attributed to Braine's and Hartnell's contact with microorganisms that naturally produce them or to random mutation in the chromosomes of Clostridium strains. Nonetheless there is an evidence that heavy metals and some pollutants may select for antibiotic-resistant bacteria, generating a constant source of them in small numbers.
Control questions:
How define sensitivity of microorganisms to antibiotics?
How defined the spectrum of antimicrobic action of a producer?
History of development of antibiotic resistance?