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This chapter introduces microbiology and basic techniques in microbiology. It gives overview of the techniques involved in culturing microorganism, including preparation and sterilization of culture media and isolation of pure cultures by aseptic techniques using the streaking method, pour-plate, spread-plate, and subculture techniques. It then explains enumeration of microorganisms by direct (direct microscopic count) and indirect methods such as standard plate count and then stains and staining techniques. By the end of the chapter methods as well as identification of bacteria through colonial characteristics, cellular morphology, biochemical procedures, serological procedures, and molecular techniques will be known. A discussion on antimicrobial susceptibility testing as well as microscope and its working mechanisms were also included.

Bacteria in non-fat dried milk (NDM) were enumerated by a method involving preliminary solubilization of the milk proteins in 0.015 N NaOH followed by centrifuging, washing in the NaOH, and microscopically examining stained smears. The method was used to enumerate bacteria in samples of NDM obtained from government surplus stocks or from local retail sources. Bacterial counts from surplus NDM ranged from 4.64 × 105 to 2.83 × 106/g (the mean and median were, respectively, 6.23 and 2.84 × 106/g). Counts from retail samples ranged from 4.48 × 105 to 2.42 × 107/g (mean and median were 5.57 and 2.85 × 106/g). The predominant bacteria in some samples were paired streptococci; other samples contained rod-shaped bacteria, some with identifiable spores. Comparison of this method with the Levowitz-Weber method indicated that it produced fewer artifacts, was applicable to NDM samples containing a wider range of bacteria, and did not require the use of the potentially carcinogenic tetrachloroethane.

Samples of skim milk and nonfat dry milk (NDM) made from it were collected, paired and tested for pyruvate concentration, [P], and Direct Microscopic count (DMC). The skim milk was tested for Standard Plate Count (SPC) and Psychrotrophic Plate Count (PPC). The geometric average DMC of skim milk was more than three times higher than that of the paired NDM samples. However, [P] of NDM was not significantly different from that of the skim milk. Although [P] of skim milk was poorly correlated with SPC and PPC, r = .31 and .26, respectively, it was relatively well correlated with DMC, r = .64. Data were widely dispersed around the regression line when [P] was ≤ 4.0 mg/L. However, [P] increased rapidly when DMCs were > 106/ml. A limit of 10 mg/L of [P] in NDM reconstituted 1:9 was chosen to represent the current U.S. Department of Agriculture Standard for DMC in NDM. This limit failed to classify about 10% of the samples correctly, assuming that each geometric mean DMC was correct. However, the probability that...

SummaryIntroduction The CellaVision Advanced Red Blood Cell (RBC) Software Application is a new software for advanced morphological analysis of RBC, which automatically performs a preliminary characterization and grouping of RBC into 21 morphological categories, including schistocytes. Upon automated classification, the software offers the possibility of reclassification of RBC by the operator. The aim of this study was to evaluate the schistocyte analysis by the CellaVision Advanced RBC Application. Methods Schistocyte counts were evaluated comparing the automated count on a CellaVision DM96, both before and after reclassification, with the reference manual microscopic method according to the ICSH criteria. Thirty-six samples of hospitalized patients and 40 samples of controls were analyzed. Results Within-run, between-run and between-observer coefficients of variation were lower when counted with the CellaVision compared to the manual microscopic count. The very high sensitivity but rather poor specificity implicates the need for reclassification by the operator, following automated analysis. After reclassification, method comparison studies revealed good agreement with the manual microscopic method, with however slightly higher values of schistocytes for the automated analysis. Conclusion The CellaVision Advanced RBC Software Application provides a sensitive and reproducible measurement of schistocytes in peripheral blood, but still requires manual revision. Furthermore, it is an easy-to-use software and an excellent teaching tool that might contribute to standardization in the investigation of schistocyte-related conditions.

In this work, we exploit a general flow cytometry technique involved in the differentiation of live and dead yeast cells for two applications in winemaking. The discrimination of yeast populations is achieved using two fluorescent dyes that measure the metabolic activity and membrane integrity of the yeast. This analytical approach is first applied for quality control of active dry yeast. Results are discussed in comparison with the Codex Oenologique International (International Oenological Codex) of the International Organisation of Vine and Wine (OIV), demonstrating that analysis using flow cytometry is a valuable alternative, given the ease of execution and the high quality of results obtained in terms of reproducibility, repeatability, and confidence interval. In the second case, we apply flow cytometry as a technique for monitoring the production of sparkling wines using the “Champenoise” method, and describe the evolution of yeast through the production process. In this case, results are directly compared with those obtained with the two methods (plate counts and direct microscopic count) listed in the OIV standards, in order to ensure a thorough understanding of the improvements related to the use of flow cytometry.

SummaryIntroduction Pentra MS CRP is a new automated hematology analyzer that can rapidly and reliably provide 5-part differential of leukocytes (5-Diff) and C-reactive protein (CRP) within approximately 3.5 min using a small volume of whole blood (35 μL). Methods We evaluated the basic performance of Pentra MS CRP and correlations with Sysmex XN-3000, manual microscopic count, and Hitachi LABOSPECT. Results Pentra MS CRP demonstrated good repeatability and linearity without any significant carryover for all parameters examined (WBC, RBC, HGB, Hct, PLT, 5-Diff, and CRP). Complete blood cell count (CBC) data examined by Pentra MS CRP correlated well with those evaluated by Sysmex XN-3000 (R ≥ 0.9880). Absolute number of NEU, LYM, and EOS also showed the good correlation (R ≥ 0.9866) between the two analyzers. The correlation with the manual microscopic count was within acceptable criteria. Furthermore, when CRP was examined in hemolyzed whole blood by Pentra MS CRP and converted to plasma concentrations according to Hct, it correlated well (R = 0.9964) with serum CRP examined by Hitachi LABOSPECT. Conclusion Pentra MS CRP is a convenient and reliable analyzer especially in the emergency unit of hospitals in which the prompt and simultaneous measurement of CBC including 5-Diff and CRP is often necessary.

Summary Introduction: The diagnosis of thrombotic microangiopathies (TMA) or disorders that may mimic their features remains difficult. Mechanical hemolytic anemia with the detection of shistocytes on the blood smear is a cornerstone finding to assess the diagnosis, but microscopic evaluation of shistocytes is still problematic with wide interobserver variations. Some of the latest generation automated blood cell counters (ABCC) propose an original quantitative approach of fragmented red cells (FRC), aiming to be equivalent to the microscopic count. This parameter has been poorly evaluated. Methods: To assess the predictive value (PV) of this test, we conducted studies comparing automated and microscopic counts of FRC/schistocytes, based on the analysis of thousands samples in four university hospitals and using the 2 ABCC currently available (Siemens ADVIA series, Sysmex XE-2100). Results: Reference range for FRC was

Summary The objective of this study was to develop a rapid quantitative detection method by triphosphate (ATP)-bioluminescence and determine the feasibility to assay total bacterial counts (TBC) in probiotic products. A useful pretreatment technique to eliminate the interference to the ATP-bioluminescence method was developed, resulting in a 10–100-fold improvement in the rapid quantitative detection method by ATP-bioluminescence. The TBC obtained by the ATP-bioluminescence rapid detection method was tested against the direct microscopic count method or the plate count method and validated on three ATP Assay Systems. The results generated by the ATP methods and the plate count method were comparable to each other when the interference due to non-microbial cell ATP and matter such as pigment were removed. This study reports the optimisation of an ATP-bioluminescence assay using a pretreatment technique to eliminate interference in order to quickly determine the viable counts of bacteria in solid and liquid probiotic products.

Objective: The objective of this study was to develop a low-cost kit for the detection of subclinical mastitis (SCM) and to check its validity, reproducibility, and efficacy at the field level. Materials and Methods: A total of 550 quarter milk samples from crossbred dairy cows were collected, of which 400 milk samples were used to validate the newly developed BLRI mastitis test (BMT) kit to justify its efficacy as an individual test kit in detecting SCM based on somatic cell count (SCC) by direct microscopic count (DMC). The efficacy of the newly developed BMT was compared with the California Mastitis Test (CMT) kit. Another 150 milk samples were subjected to SCC determined by DMC and DCC (De Laval cell counter®) categorized by CMT and BMT scores. Results: A SCM test kit, namely, BMT kit was successfully developed in this study. The percentage accuracy of CMT and BMT were 76.75% and 75.75%; sensitivity 69.36% and 67.56%; specificity 85.95% and 85.85%; positive predictive value 86.03% and 85.71%; negative predictive value 69.23% and 68%, respectively. A p value of 0.001 was found for both CMT and BMT. However, CMT and BMT had no significant difference in sensitivity (p = 0.778). Average SCCs (cells/ml) determined by DCC and DMC, respectively, were mostly corresponded to the SCC ranges of both CMT and BMT scores. Conclusion: The newly developed BMT kit is an independent, cheap, farmer-friendly, first country made, and reliable SCM diagnostic test kit that can be used at field condition. [J Adv Vet Anim Res 2019; 6(3.000): 425-430]

Summary Introduction: Performance of the Celltac F haematology analyser (MEK-8222) which provides 22 parameters, including a 5-part differential, was compared with the Sysmex XE-2100. Methods: 242 EDTA samples were investigated. Differential results from both instruments were compared with the reference microscopic count. Flagging performance was compared with cell morphology seen in the blood films. Results: Precision met or exceeded manufacturer’s specifications, carryover was minimal (≤1.37%) and linearity was excellent (R ≥ 0.99). Results were stable for at least 8 h at room temperature and for 24 h at 4 °C. Comparisons were excellent for white blood cells, red cell count, Hb, HCT and platelets (R ≥ 0.98). All other red cell and platelet parameters showed good correlation with the XE-2100 (R ≥ 0.93) except for mean cell haemoglobin concentration. The differential was comparable to the XE-2100 for neutrophils, lymphocytes and eosinophils and acceptable for monocytes. Correlation of automated differentials with manual reference counts and the efficiency of flagging of blasts, immature granulocytes and platelet clumps were similar for both instruments. Celltac F demonstrated better efficiency for atypical lymphocyte and platelet clumps. Conclusion: The Celltac F shows broadly comparable analytical performance to the XE-2100 for the parameters assessed. The Celltac F is recommendable for medium-sized laboratories or as a back-up instrument in larger laboratories.

Periodontal disease is an inflammatory reaction to the bacteria in dental plaque. The present study compared the prevalence of periodontal disease in patients using as a diagnostic either probing depth measurements, an inflammatory marker such as numbers of white blood cells in plaque samples, or microbiological markers such as the microscopic count and the benzoyl-DL-arginine naphthylamide (BANA) test.Teeth with the most inflammation and/or deepest pockets in each quadrant were probed and subgingival plaque was sampled from 1,043 consecutive new patients enrolled in a private practice. Multivariate "diagnostic" models were developed based upon the probing depth (general linear models), percentage of white blood cell-positive and percentage of BANA-positive plaques (logistic regression models) to determine the prevalence of patients with periodontal disease.Plaque samples were removed from 3,694 sites. Fifty-two percent of sampled pockets were4 mm; 49% of sites were inflamed, using the presence of white blood cells, and 28% were infected using the BANA test. Diagnostic models were highly significant at P0.0001. The white blood cell model was the most parsimonious as demonstrated by the lowest Akaike information criteria statistic and had the highest receiver operator characteristic (ROC) curve relative to the probing depth and BANA models.Periodontal disease can be diagnosed chairside by the presence of white blood cells in plaque samples, a finding that reflects the inflammatory nature of the disease process. This approach would reduce the misclassification of subjects as having periodontal disease (130 patients in the present study who had pockets) but minimal evidence of an inflammatory response.

This study investigated a microfluidic chip-based system (on-chip flow cytometry) for quantification of bacteria both in culture and in environmental samples. Bacterial numbers determined by this technique were similar to those obtained by direct microscopic count. The time required for this on-chip flow cytometry was only 30 min per 6 samples.

Minimum inhibitory concentration (MIC) isobolograms of fresh garlic extract (FGE) and NaCl against Staphylococcus aureus test strains ATCC 25923 and ATCC 6538 in Brain Heart Infusion broth (BHIB) and Pork Sausage broth (PSB) were constructed. Additive and synergistic relationships were observed in BHIB and PSB against the test strains, respectively. Action of indirect antimicrobic ingredients in PSB was used to explain the synergistic relationship of FGE and NaCl established. The Fractional Inhibitory Concentration (FIC) indices of the agents validated the antimicrobic relationships established in the MIC isobolograms. An Iodonitrotetrazolium-Direct Microscopic Count (INT-DMC) method was used to measure staphylococcal cell count reductions in both broth systems containing concentrations of FGE and NaCl typically used in sausage production. Garlic incorporation of ≥1.20 (v/v) together with minimum levels of NaCl (

The diversity of bacterial populations developed in the surface layer (0–0.25 m depth) of volcanic mudflow deposits from the Mt. Pinatubo volcano (the Philippines) was investigated using quinone profiling. Samples were collected from sites (named N and S1) that had been hit repeatedly by mudflows during successive rainy seasons after the violent eruption of 1991 and also from sites (F1 and F3) covered by mudflow in 1991 but with no deposition in following years. The total microscopic count ranged from 10 8 g −1 (N and S1 sites) to 3.9×10 9 g −1 (site F3). In the N sample only three quinone species were detected, while the quinone profiles of samples from sites S3 and F3 showed higher diversity. Tetrahydrogenated menaquinone with eight isoprenoid units [MK-8 (H 4 )] was the predominant quinone species in the sample from site N, while MK-8, MK-8 (H 2 ), MK-8 (H 4 ), MK-9 (H 4 ) and MK-9 (H 8 ) were found as major quinones in the sample from site F3. Because these MK species are known to be the major respiratory quinones of the Actinobacteria , this bacterial group is expected to predominate in the land with primary vegetation recovery following the impact of the volcanic mudflow.

Mikroskobik yöntemler, mikrobiyel hücrelerin varlığının belirlenmesinde çok hızlı yöntemlerdir. Standart kültürel yöntemler ile karşılaştırıldığında canlı ve ölü hücrelerin beraberce sayılması gibi bir dezavantajı bulunmakla beraber, hammaddenin mikrobiyel durumu hakkında bilgi vermesi açısından avantaj da taşımaktadır. DEFT, basit direk mikroskobik sayım yöntemlerinden yaklaşık 100 kat daha duyarlı ve sonuçları kültürel sayım ile kıyaslanabilecek kadar doğru sonuçlar veren bir yöntemdir. DEFT yöntemi canlı ve ölü hücreler arasındaki ayrımı sağlamaktadır. Fluorochrome işaretli antikorların kullanımı ile belirli mikroorganizmaların tür düzeyinde tespiti de mümkündür.

Enzyme-linked immunospot (ELISPOT) is a sensitive technique for the detection of cytokines released by immune cells. The technique is well established and correlates closely with the enzyme-linked immunosorbent assay (ELISA) technique. Here, we introduce an integrated imaging-analysis system to quantitate the spots formed by the ELISPOT assay. The spots can be easily counted when the background is clear and the spots are few. However, when the number of spots increases to >100, this task becomes challenging and time-consuming. To minimize time and standardize the counting procedure, we have used a digital camera linked to a computer system for reading the plates. In general, the computer is able to count more spots and has a smaller standard deviation when compared with the manual microscopic count. This integrated system is commercially available, and we believe that this method is objective, timesaving and consistent in routine ELISPOT assay counting, especially when numerous spots are present.

An evaluation of the flow cytometric procedure for reticulocyte counting was performed, together with a comparison to a reference manual counting method. An automated reticulocyte analyser, the Sysmex R-3000™ (Flow cytometric auramine O methods) was used to enumerate the absolute reticulocyte counts and the percentage of reticulocytes in blood obtained from rats, dogs and monkeys. Simultaneously, a maturation distribution was obtained by subdividing the reticulocytes into three maturation classes according to their fluorescence intensity. The reticulocyte counts and the maturation distributions can be determined rapidly and easily. The coefficients of variation (CV) for the within-day precision study in the reticulocyte counts of the three species were found to be 9.7%–20.5% for the micro-scopic method and 4.4%–9.4% for the flow cytometric method. A very good correlation was observed between the microscopic count and flow cytometric methods (r=0.958). In conclusion, because of high reproducibility and good agreement with the manual counting method for these species, flow cytometric analysis of reticulocytes is an attractive alternative procedure to micro-scopic enumeration in toxicological studies.

A description is given of a pre-treatment for milk samples which allows it to be filtered through 0.2 μm pore-size membrane filters without reducing the initial number of bacteria. The efficiency of the method was demonstrated in milk samples inoculated with Escherichia coli O25:H-42, a food-borne pathogen, by comparison with a control in tryptone soya broth (TSB). No significant differences were found between direct microscopic counts in milk samples and controls in TSB.

Geosmin has often been associated with off-flavor problems in drinking water with Anabaena sp. as the major producer. Rapid on-site detection of geosmin-producers as well as geosmin is important for a timely management response to potential off-flavor events. In this study, quantitative polymerase chain reaction (qPCR) methods were developed to detect the levels of Anabaena sp. and geosmin, respectively, by designing two PCR primer sets to quantify the rpoC1 gene (ARG) and geosmin synthase one (GSG) in Anabaena sp. in freshwater systems. The ARG density determined by qPCR assay is highly related to microscopic cell count (r(2) = 0.726, p < 0.001), and the limit of detection (LOD) and limit of quantification (LOQ) of the qPCR method were 0.02 pg and 0.2 pg of DNA, respectively. At the same time, the relationship between geosmin concentrations measured by gas chromatography-mass spectrometry (GC-MS) and GSG copies was also established (r(2) = 0.742, p < 0.001) with similar LOD and LOQ values. Using the two qPCR protocols, we succeeded in measuring different levels of ARG and GSG copies in different freshwater systems with high incidence environmental substrata and diverse ecological conditions, showing that the methods developed could be applied for environmental monitoring. Moreover, comparing to the microscopic count and GC-MS analytical methods, the qPCR methods can reduce the time-to-results from several days to a few hours and require considerably less traditional algal identification and taxonomic expertise.

Variations in the structure of the protozoan community living in activated sludge were studied in relation with the organic loading rate. 84 samples were taken over a 14 month period. Sludge and wastewaters were subjected to microscopic and physico-chemical analyses. The organic loading rate corresponding to each microscopic count was determined as the average of the values calculated for the 2 days previous to the count. Ciliate species abundance and specific diversity of ciliates were strongly related to the organic loading rate. Data were compared to three other facilities. Ciliate diversity decreased as the organic loading rate increased. This relationship could be modified if solids in suspension surpassed 4000 MLVSS mg/l or if volumetric loading rates were higher than 800 g BOD 5 /m 3 day. An easy, fast diagnostic test to estimate the organic loading rate in less than 5 days and to obtain information on plant performance is proposed.

This procedure provides a reproducible method to collect cells on 10-mm-diameter Zeta-Probe blotting membrane discs. This method allows for a defined number of cells to be available for studies with intact tissue in cell-tissue electrofusion protocols. In addition, this procedure can be used to rapidly and efficiently remove nonadherent cells from suspension cultures. The number of cells placed on these electrostatically charged membranes can be quantitated spectrofluorometrically or by microscopic count. Once the cells are placed on the Zeta-Probe they can be moved or inverted without falling off. In addition, the discs can remain at room temperature for 3 to 5 h without a significant change in the number of layered cells or an adverse effect on the fusion process. The ability to control the number of cells placed in contact with tissue will allow for the further development of cell-tissue electrofusion.

The effectiveness of a dispersion and elutriation method for sampling soil microorganisms, in which the soil was dispersed with an anionic detergent and a chelating ion-exchange resin followed by separation of the microorganisms from soil particles by elutriation in an upwardly flowing liquid column, was tested using a bacterial cell suspension, a clay soil and a sandy soil. On theoretical considerations alone, the relationship between elutriation flow rate and the sedimentation velocity of particles suggested that complete separation of microorganisms from soil particles was impracticable. This was because separation of non-filamentous microorganisms with minimum contamination by other soil particles required excessively slow elutriation flow rates. A compromise situation in which a lower elutriate purity was off-set by increased elutriation flow rate was therefore tested. A pure suspension of Bacillus subtilis cells was not elutriated at the rate predicted by Stoke's law. This was attributed to non-uniform liquid flow within the elutriator which effectively retarded the upward movement of cells in the elutriators. When tested with soil, between 70 and 80% of the microscopically-counted cells and between 50 and 60% of the ATP were recovered in 41 of clutriate. Problems in completely dissociating microorganisms from soil particles made it impossible to produce an extract containing exclusively microorganisms. Nevertheless, the ratio of biomass (direct microscopic count or ATP) to the weight of soil particles was increased, when compared to that in the soil at the outset, by factors of 2.3 and 4.3 for the clay and the sandy soils, respectively, when determined by direct microscopic counts and by factors of 1.8 and 3 for the same soils when determined by ATP contents. The differences in concentration factor between the soils were attributed to the relative ease with which the two soils were dispersed. It was concluded that the major problems that need to be overcome if this technique is to be useful in sampling microorganims from soil are: (1) complete dispersion of the soil and dissociation of microorganisms from soil particles; (2) maintaining uniform clutriation flow; and (3) concentrating the dilute elutriate sample produced.

Bacteriophage are viruses of bacteria. An enormous number of bacteriophages are found the world over, on the order of 1030 or more. This article first presents an overview of techniques employed to characterize phage existence in natural environments, including by direct microscopic count, viable count, and various molecular means of phage in situ characterization. Then it provides an overview of the science of phage ecology. This overview is followed by four sections in which four subdisciplines of phage ecology – phage organismal ecology, phage population ecology, phage community ecology, and phage ecosystem ecology – are discussed in detail. These represent, respectively, (1) the impact of phage growth parameters (such as latent period, burst size, and virion resistance to decay) on phage ecological success, (2) phage population growth and intraspecific interaction, (3) the phage impact especially on bacterial communities, and (4) the phage impact on environmental energy flow and nutrient cycling. Note that these various means of looking at phage ecology build upon one another.

In 37 samples from the river Warnow in the course of one annual variation the seston particles were counted in the inverted microscope in three size classes: A 25μm. Parallel to that, counting was performed with the electronic particle counting device Picoscale 4, which is used for the counting of blood cells, in the setting WBC for leucocytes with a nozzle 200 μm in diameter, so that the setting approximately corresponded to the size range B of the microscopic count. The microscopic particle numbers per ml were 800,000 … 5,200,000 for A, 10,000 … 96,000 for B and 2,000 … 21,000 for C. In principle, there is a correlation between microscopic and electronic results for the particles B and C. The regression analysis, however, produces a considerable residual scattering because of the morphological variety of the particles and its changes in the course of the seasonal change of mass. Therefore, the conclusion of the particle density from results of electronic counting is connected with considerable indistinctness, so that the electronic counting can be used only as a supplementary method for the estimation of the density of suspended matter.

As avaliações quantitativas e qualitativas das células presentes no leite são empregadas para a compreensão de diversos processos, fisiológicos ou não da glândula mamária. Apesar disso, as citações da literatura são conflitantes quanto às búfalas sadias e, para elucidar essa questão, avaliou-se a celularidade do leite em 108 amostras de quartos mamários de búfalas hígidas em lactação, submetendo-as à contagem de células somáticas (CCS), automática e por microscopia óptica, além de estabelecer-se a predominância celular empregando duas técnicas (Prescott e Breed e citocentrifugação). Os resultados das CCS do leite, por técnicas microscópica e automática foram semelhantes, mas a predominância celular analisada por microscopia diferiu em valores relativos. Foram identificadas mais células mononucleares nas lâminas de suspensão celular citocentrifugada, e mais leucócitos polimorfonucleares na técnica de Prescott e Breed. A lâmina de citocentrifugação permitiu melhor avaliação da morfologia celular sendo identificados 61,1% de monócitos e macrófagos; 32,9% de neutrófilos; 5,3% de linfócitos e 0,7% de eosinófilos. Os fagócitos mononucleares apresentaram uma acentuada plasticidade de suas estruturas, com variados padrões morfológicos.

With an objective to find microbial quality of the market milk available in Kathmandu valley, this study was conducted in the Microbiology Laboratory, Himalayan College of Agriculture Science and Technology (HICAST). Different milk brands were collected from different outlets in the valley and analyzed for microbial quality. The samples were cultured using the standard microbiological techniques. Almost all the samples showed the presence of bacterial growth including coliform bacteria. The bacterial isolates were of 7 different genera. E. coli was the most frequently isolated organism (92%) followed by coagulase negative Staphylococci (CONS) (24%). Other organisms isolated were Bacillus species, Staphylococcus aureus, Streptococcus species, Salmonella species, Shigella species. The culture results were similar to direct microscopic count results and showed that bacterial contamination in some of the samples was too numerous to be counted. Thus, it was concluded that the market milk available in Kathmandu is contaminated with various types of microorganisms and does not meet the required standard. The dairy industries should work seriously to maintain the standards of their products. * Integrated Bioscientific Research Group (BIOINT), Kupondol, Lalitpur, Nepal. ** Himalayan College of Agriculture Science and Technology (HICAST), Gatthaghar, Bhaktapur, Nepal. Address for correspondence : Charu Arjyal Integrated Bioscientific Research Group (BIOINT), Kupondol, Lalitpur, Nepal. Email: charuarjyal@hotmail.com Key Words: Microbiology, Contamination, Milk, Quality, Coliform.

Introduction: The CellaVision Advanced Red Blood Cell (RBC) Software Application is a new software for advanced morphological analysis of RBC which automatically performs a preliminary characterization and grouping of RBC into 21 morphological categories, including schistocytes. Upon automated classification, the software offers the possibility of reclassification of RBC by the operator. The aim of this study was to evaluate the schistocyte analysis by the CellaVision Advanced RBC Application. Methods: Schistocyte counts were evaluated comparing the automated count on a CellaVisionTM DM96, both before and after reclassification, with the reference manual microscopic method. 76 samples of both hospitalized patients and healthy donors were analyzed. Results: Within-run, between-run and between-observer coefficients of variation were lower when counted with the CellaVision compared to the manual microscopic count. The very high sensitivity but rather poor specificity implicates the need for reclassification by the operator, following automated analysis. After reclassification, method comparison studies revealed good agreement with the manual microscopic method, with however slightly higher values of schistocytes for the automated analysis. Conclusion: The CellaVision Advanced RBC Software Application provides a sensitive, accurate and reproducible measurement of schistocytes in peripheral blood. Furthermore, it is an easy-to-use software and an excellent teaching tool that might contribute to standardization in the investigation of schistocyte-related conditions. Disclosures No relevant conflicts of interest to declare.

A standard method for enumerating marine bacteria from a water sample is to grow them on nutrient plates and count colonies. In low nutrient aquatic habitats, however, the number of bacterial colonies that can be grown using standard plating methods is frequently from one to four orders of magnitude fewer than the number of cells observed by direct microscopic count (Jannasch and Jones 1959). This well-documented observation, dubbed the “great plate count anomaly” (Staley and Konopka 1985), has led to much speculation about the identity, physiological ability and metabolic status of the observed but apparently uncultivatible microbes. These are well-defined questions that have nevertheless gone unanswered since the late 1800’s (Roszak and Colwell 1987).

This paper describes a comparison between the microscopic brilliant cresyl blue and the flow cytometric thiazol orange (FACScan) and auramine O (Sysmex) methods for enumeration of reticulocytes. The mean intra-assay coefficients of variation for the microscopic, FACScan and Sysmex methods were established to be 33.9 and 5% respectively. A rather poor correlation was observed between the microscopic count and both flow cytometric methods (FACScan r = 0.61; Sysmex r = 0.57). However, the correlation between the flow cytometric methods was satisfactory (r = 0.79). Reference ranges for the reticulocyte count, corresponding with the 2.5th and 97.5th percentile, were determined for the microscopic (8-30/1000), FACScan (11-27/1000) and Sysmex (8-18/1000) methods. Sysmex R-3000 methodology definitely revealed the lowest and narrowest reference range. In conclusion, because of higher reproducibility, flow cytometric analysis of reticulocytes is an attractive alternative procedure for the microscopic enumeration method.

Previous cultural microbiology and fluorescence labelling studies indicate an ecological basis for the relationship between Streptococcus mutans and human dental caries. The present study further investigated the basis of this postulated relationship by sampling gingival margin plaque at sites more or less stagnant and, therefore, more or less caries prone in relation to the contact area. Six hundred and thirty-two samples were obtained from 180 teeth in 74 children, comprising gingival margin approxinial plaque from embrasure (away), to the side of, over and below the contact area. These were processed by indirect inimunofluorescence using an S. mutans polyclonal antiserum. While S. mutans was not detectable in many sites, in spite of high sensitivity and specificity of the antiserum, there was a significant tendency ( P < 0.05) for the proportional direct (labelled) microscopic count to increase apical to the contact area. Keywords: Streptococcus mutans ; Caries; Ecology.

Electron transport was demonstrated cytochemically in soil micro-organisms. After counterstaining, enzymically active and inactive cells were distinguishable. The number of active micro-organisms detected by this method ranged from ca. 11 — > 46% of the total microscopic count in several soils. The technique was also applied successfully to algae from soil crusts, micro-colonies of microorganisms removed intact from the surface of soil crumbs and to micro-organisms associated with plant roots.

About 106 of total bacteria (direct microscopic count) or heterotrophic bacteria (colony count with Medium 2216E) and about 104 of petroleumlytic bacteria (oil-oxidizing bacteria) were enumerated per square centimeter of the surface of petroleum globules suspended in the topmost 10 meters of water from a station (22°00.2′N, 125°51.9′E) in the western North Pacific central water, where about 107 of total bacteria, about 103 of heterotrophic bacteria and about 10 of petroleumlytic bacteria were enumerated per liter of seawater.

The automated test for pyruvate concentration, [P], was evaluated as a substitute for the direct microscopic count (DMC) in determining quality of manufacturing grade milk. Samples of manufacturing grade milk from producers and tank trucks as well as skim milk were collected at a large milk drying plant. Each sample was tested immediately for standard plate count (SPC), psychrotrophic plate count (PPC) and DMC. Portions of milk were heated to 85°C for 10 min to stabilize [P] before being returned to the laboratory for analysis of initial pyruvate concentration, [IP]. Unheated samples stored at 4°C were analyzed for [P] and DMC daily for up to 120 h. [IP] was a good indicator of PPC in milk from individual producers (r=0.81). However, [IP] was not highly correlated (r=0.26) with PPC in skim milk samples which were characteristically high in SPC and homogeneous in PPC. With skim milk, [IP] was more highly and significantly correlated with initial DMC (r=0.61) than with initial SPC (r=0.31) or initial PPC (r=0.26). [IP] was a good indicator of [P] in stored fluid samples until counts exceeded about 10

Taxonomic screening of subgingival plaque organisms with various enzyme assays have shown that Treponema denticola, Bacteroides gingivalis and an unspeciated Capnocytophaga species possess a trypsin-like enzyme (TLE) that can be detected by the hydrolysis of N-benzoyl-DL-arginine-2-naphthylamide (BANA). As these organisms can be considered to be periodontopathic, it was of interest to determine whether this BANA hydrolyzing enzyme could be detected directly in subgingival plaque samples. Subgingival plaque samples were collected from single sites of known pocket depth, and after dispersal by vortexing, aliquots were incubated overnight with BANA and were counted microscopically. The color reactions were developed with fast garnet, read by the eye and classified as positive (red to red-orange), negative (yellow) and questionable. In the BANA-positive plaques, the spirochetes averaged 43% of the microscopic count, whereas in the BANA negative plaques the spirochetes averaged 8% of the microscopic count. The average pocket depth of BANA-positive plaques was 6.7 mm, whereas the average pocket depth of BANA-negative plaques was 4.5 mm. When both of these parameters were combined, the presence of a positive BANA reaction was usually indicative of subgingival plaques containing greater than 34% spirochetes removed from sites that had probing depths of 7 mm or more. Seventy-one per cent of the plaques removed from untreated periodontal patients were BANA-positive, while only 8% of the plaques removed from successfully treated patients seen at maintenance recall visits were BANA-positive.(ABSTRACT TRUNCATED AT 250 WORDS)

Annual fluctuations of bacterial numbers were surveyed at four stations in Lake Biwa between 1981-1982. The total count obtained by acridine orange direct microscopic count method changed in the order of 105-106cells per ml at St. Ie (sited in the pelagic area of the north basin), and 106-107cells per ml at three stations in the south basin. The percentage of the viable count to the total count (V/T ratio) ranged from 0.009 to 1.81%, and the ratio tended to be higher in more eutrophic water. Generally, at St. Ie, the vertical profiles of both total and viable counts clearly correlated with those of chlorophyll-a, pH and dissolved O2 (% saturation); however, hypolimnetic O2 depletion had no effect on bacterial numbers. Rank correlation analysis for the samples of epilimnion also showed significant correlations (p

Summary Acetomicrobium flavidum gen. nov., sp. nov. was isolated from a thermophilic, anaerobic sewage sludge digester operated at 60 °c. The Gram-negative, highly motile, non-sporeforming rods form 2 mol acetic acid, 2 mol CO 2 and 4 mol H 2 from 1 mol hexose. They represent at least 25% of the total population (microscopic count) of the fermenting sludge.

In this report over 400 subgingival plaque samples taken from over 110 patients were examined microscopically and culturally for 30 bacterial parameters. The patients could be placed into six disease categories based upon clinical criteria. The bacterial profile of each clinical category was generally distinctive of that category. Periodontal patients who had been successfully treated and maintained had plaques that were populated by significantly higher proportions of Streptococcus sanguis, Actinomyces viscosus, A. odontolyticus and S. mutans and significantly lower proportions of B. gingivalis and spirochetes compared to the five untreated disease categories. The spirochetes were the overwhelming microbial type in the plaques of adult periodontitis (AP) patients, averaging about 45% of the microscopic count. The bacteriological results could not distinguish between ADA Type III and IV periodontitis, suggesting that the same type of infection was occurring in an active site in any AP patient. The patients designated as early onset periodontitis (EOP) differed from the other patients by their relative youth and by their significantly higher proportions of Bacteroides gingivalis and/or B. intermedius. Two types of EOP were recognized in which the most diseased variant was characterized by having an average of 49% spirochetes in the plaque. Four localized juvenile periodontitis (LJP) patients were notable in not having detectable A. actinomycetemcomitans. The data indicate that the various types of periodontitis, with the possible exception of LJP are specific anaerobic infections involving spirochetes and to a lesser extent B. gingivalis and B. intermedius.

With three natural soils from Western and Central Norway it has been shown that by homogenization and fractionated centrifugations, two fractions could be obtained, one containing 50–80% of the bacteria, and the other all the fungi together with soil debris and the rest of the bacteria. When the separation was carried out in the cold and lasting less than 3–4 h, the fractions if used immediately had constant respiratory rates for 1–2 h. The sum of the rates agreed well with that of an unfractionated soil homogenate, making it possible to calculate the ratio between bacterial and fungal respiration. Assuming that the much higher respiration in a homogenate than in intact soil is due to inhibitory conditions in the latter and that these can be analysed by separate experiments, the technique will permit estimates of the ratio between bacterial and fungal respiration in in situ soil under given conditions. The soils used had very great differences between plate and microscopic count values for bacteria. When based on plate counts, the calculated bacterial respiratory intensities became impossibly high, while they were within biologically possible limits when microscopic count values were used. The respiratory intensities for fungi when calculated from hyphal lengths and diameter measurements, were biologically possible.

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The results of the Feulgen-DNA-color optical density and score, the California mastitis test (CMT) score, viscosity developed in milk samples by addition of equal volume of CMT reagent, the catalase test, the (direct microscopic count bf leucocytes, and the proportion of live leucocytes were compared using fresh, quarter, and bucket milk samples and 1 to 2 day old bulk milk samples. The Feulgen-DNA-color gave. good correlations with all the tests in all types of samples, except with the CMT of bulk samples. The CMT score and viscosity gave good correlations with all the tests on fresh milk samples and insignificant correlation with stored milk samples. The catalase test gave good correlations with all other tests in fresh samples and low but significant correlations with stored samples. Quarter and bucket milk samples contained 71% and 74% live leucocytes and bulk milk samples contained 42% live leucocytes. Holding of milk at 5 C for 5 days did not significantly change the Feulgen-DNA-color and catalase activity, slightly increased total leucocyte counts, but gradually decreased the CMT score, the viscosity, and the proportion of live leucocytes. The Feulgcn-DNA-color of milk samples containing few millions of leucocytes and the direct microscopic count of total, leucocytes gave poor reproducibility.

Summary The standard plate count (SPC-32 C) and the direct microscopic count (DMC) of samples of commercially pasteurized milk inoculated with pure cultures of actively growing (18–24 hr growth) bacteria commonly found in milk were compared. Four staining procedures for DMC were used: (a) Levowitz-Weber's methylene blue stain; (b) a modified Levowitz-Weber stain incorporating basic fuchsin; (c) alcoholic-acetic acid fixation followed by periodic acid-bisulfite treatment and staining with pH4 toluidine blue; and (d) alcoholic-acetic acid fixation and staining with pH4 toluidine blue. Counting was standardized by the use of a geometrical pattern. Correlations between SPC and each DMC procedure, or among the DMC procedures were little influenced by the number of microscopic fields counted, their location on the smear or the definition of “clumping” used. Correlations were influenced by the type of bacterial culture inoculated in milk and by the staining procedure. Precision of DMC was shown to be independent from the staining procedure, but varied directly with the number of cells per field and inversely with the square root of the number of fields counted.

The bacteriological quality of manufacturing-grade milk is very similar to that marketed a decade earlier when bulk tanks first came into general use. Milk grading programs usually relied on reduction tests. These tests indicated that most milk supplies were good quality. Based on the Standard Plate Count, data is presented that show approximately one-third of the samples tested, in 1969–70 and in 1957–59, exhibit counts Dairy farmers learned they could substitute cooling for cleaning because psychrotrophic bacteria predominated the microflora of most high count bulk milk. These bacteria do not readily reduce resazurin and methylene blue. Psychrotrophs also tend to grow in clumps preventing an accurate evaluation of milk quality using the Direct Microscopic Count (DMC). USDA uses the DMC to test check manufacturing plant's milk supplies. Laboratories are recognizing the value of plating procedures, including the Plate Loop Count, to determine milk quality. Manufacturing-grade milk must be evaluated with a plating procedure before progress can be made in milk quality improvement. One grade of milk is far from being a reality if present levels of manufacturing-grade milk quality are considered.

Standard microbiological tests, Standard Plate Count, direct microscopic count, methylene blue reduction test, and several variations of the resazurin reduction test were correlated with fieldmen's ratings of sanitary condition of milking area, milk house, and milking utensils. Correlation coefficients were low, in general, approximately 0.2. The effect of different production facilities and practices on these correlations was variable. Results suggest that bacterial tests and fieldmen's inspection be used concurrently since they appear to measure different sanitary factors on the producing farms.