Commonly used Techniques for the Primary Culturing and Isolation of Clinically Important Anaerobic Bacteria
Lorraine S. Gall, Phyllis E. Riely in Manual for the Determination of the Clinical Role of Anaerobic Microbiology, 2018
The ideal technique for the primary culturing of anaerobes will be simple: protect the culture from oxygen in all phases of the technique, encourage good growth of all clinically important anaerobes in a reasonably short time while discouraging growth of contaminants, and not be too costly. This chapter considers these factors in the discussion of each procedure. The roll or streak tube technique requires the use of prereduced anaerobically sterilized media (PRAS), which refers to media that are sterilized in a reduced state and remain reduced continuously throughout the inoculation procedure. PRAS is available commercially from several well-known media supply houses or it may be prepared in the individual laboratory. The selected PRAS broth and the freshly poured agar plates stored in the chamber prior to use are employed for inoculation. The maintenance of anaerobic conditions in the chamber allows the grinding of tissues and homogenization of specimens or other preparative procedures to be made without exposure to oxygen.
Negative Stain Em
Erskine L. Palmer, Mary Lane Martin in Electron Microscopy in Viral Diagnosis, 2019
The technique of negative staining, developed by S. Brenner and R. W. Home in 1959, utilizes an electron opaque substance to surround the virus or other biological structure, giving contrast between the electron-lucent biological material and the background against which it is viewed. There are many techniques of negative stain electron microscopy, but there is no universally accepted method for grid preparation. The negative stain not only surrounds virus particle but penetrates between the viral structural units and even into the interior of the virus to delineate morphological features. Prefixation of liquid preparations of some viruses with glutaraldehyde prior to negative staining can be done to accentuate features without distorting viral stracture. The Formvar film is floated off of the agar block onto surface of a negative stain contained in a small petri dish. Three-dimensional viral stracture can sometimes be delineated by pseudoreplica technique when particles are oriented so that the view is directed toward the inside of tubular structures.
Identification of Microbes
Susan Isaac, David Jennings in Microbial Culture, 2020
An organism can be isolated from the environment and its characteristics compared with those of others. A microbe can thus be assigned to a particular genus and species (identification). All aspects of microbiology involve the use of microscopy. Microscopy also allows the study of the patterns of differentiation which occur throughout growth and development on different substrates, and also reactions of cells to added chemicals and to the presence of other microbes. Research microscopes may have many attachments and facilities so that specific local instructions will be required before any attempt to use these instruments. However, a simple microscope can give an excellent view of specimens, provided that it is set up appropriately. For some work in a microbiology laboratory the use of a stereo (or dissecting) microscope is ideal for examining the surfaces or margins of colonies (particularly fungal) growing on the surface of agar or on natural substrates.
Group B streptococcus colonisation in pregnant women at Dr. George Mukhari Hospital, South Africa
Published in Southern African Journal of Infectious Diseases, 2016
MC Monyama, JY Bolukaoto, MO Chukwu, MRB Maloba, SR Moyo, RT Mavenyengwa, M Nchabeleng, SL Lebelo
The aim of the study was to estimate group B streptococcus (GBS) colonisation in pregnant mothers using selective enrichment broth and solid media for culturing GBS. Vaginal and rectal swabs were collected from 413 pregnant women for GBS culture at recruitment stage. Direct plating and enrichment broth culture methods were compared by using the same swab samples. The swabs were cultured on colistin nalidixic agar (CNA) plate and incubated at 37°C and examined after 18-24 h. The samples which were culture negative on a CNA agar plate were then inoculated into a Todd-Hewitt enrichment broth to recover any GBS present that was not recovered on the solid agar. With the CNA agar plate, the samples were cultured separately to enable identification of colonised sites such as vaginal sites or rectal sites. Rectal and vaginal swabs were inoculated into Todd-Hewitt enrichment broth at the same time in the same tube. The GBS colonisation rate in pregnant women was 30.9% (128/413). The CNA agar plate recovered 45.3% (58/128) of the GBS isolates, whereas 54.7% (70/128) isolates were recovered from Todd-Hewitt broth. Pregnant women of various ages were found to be at risk of GBS colonisation. The colonisation rate was however highest among women of 25–29 age groups as compared with other age groups. Detection of group B streptococcus improved when both rectal and vaginal swabs were collected for laboratory analysis. The simultaneous use of Todd-Hewitt broth and CNA plate also improved the yield of group B streptococcus.
Efficacy of Topical Ofloxacin 0.3 % Administration on Conjunctival Bacterial Flora in Diabetic Patients Undergoing Intravitreal Injections
Published in Seminars in Ophthalmology, 2017
Panagiotis Plotas, Olga E. Makri, Ilias Georgalas, Nikolaos Pharmakakis, Apostolos Vantarakis, Constantine D. Georgakopoulos
Purpose: This prospective, randomized case series study aims to evaluate the efficacy of ofloxacin 0.3% eye drops in eradication of conjunctival bacterial flora in diabetic patients undergoing intravitreal injections (IVI). Methods: Ninety-two diabetic patients (92 eyes) scheduled to undergo intravitreal injection of ranibizumab due to diabetic macular edema were enrolled in the study. Patients were randomly assigned to three different groups. Group 1 (n=32) received ofloxacin eye drops the day before before IVI (four times); patients in Group 2 (n=29) were administered ofloxacin one hour before IVI (every 15 minutes), while Group 3 (n=31) comprised patients that received combined administration of ofloxacin both one day and one hour before IVI (eight doses). Samples were collected from the injection site before and after antibiotic administration. Culture results from BACTEC broth and positive cultures in blood agar and Sabouraud’s dextrose agar plates were measured. Results: In Group 1, BACTEC broth positive cultures decreased from 84.4% at baseline to 50% after ofloxacin administration (p=0.007), and blood agar positive cultures reduced from 65.63% to 34.38% (p=0.02). In Group 2, positive cultures significantly decreased in BACTEC broth (from 79.3% at baseline to 48.28%; p=0.027) and in blood agar (from 68.97% to 37.13%; p=0.034). In Group 3, positive cultures decreased from 77.42% at baseline to 32.26% (p=0.0008) and from 58.06% at baseline to 22.58% (p=0.009) in BACTEC broth and blood agar, respectively. No microorganisms were isolated from Sabouraud’s dextrose agar plates. Conclusions: The combined one day/one hour (eight doses) ofloxacin administration in diabetic patients is extremely effective in reducing conjunctival bacterial flora. The application of topical ofloxacin for one day or one hour before IVI is also significantly effective.
A Diffusion Model for Studying the Drug Release from Semisolid Dosage Forms I. Methodology Using Agar Gel as Diffusion Medium
Published in Drug Development and Industrial Pharmacy, 1986
Hung Won Jun, Samir M. Bayoumi
Several ointment bases containing three sulfonamides widely different in their physicochemical properties were evaluated for their drug release properties using agar gel as the drug acceptor phase. The drug release/diffusion model was created by making an empty cylindrical core in the center of agar gel plate which was filled with ointment containing the drugs. The results showed that a linear correlation existed when the amount of drug released from each ointment was plotted against the logarithmic time. The solubility of drugs in the base and partition properties into the agar medium played the major role for the release of drugs. The effect of temperature on the diffusion into the acceptor phase was also recorded.