Improved bioluminescent assay of somatic cell counts in raw milk

2008 ◽  
Vol 75 (3) ◽  
pp. 279-283 ◽  
Author(s):  
Valery G Frundzhyan ◽  
Inna M Parkhomenko ◽  
Lubov Y Brovko ◽  
Natalia N Ugarova
Keyword(s):  
Somatic Cell ◽  
Somatic Cells ◽  
Raw Milk ◽  
Superior Performance ◽  
Bacterial Cells ◽  
Bioluminescent Assay ◽  
Direct Microscopy ◽  
Cell Counts ◽  
Simple Protocol

Somatic cell count (SCC) in milk is considered to be a valuable indicator of cow mastitis. For assessment of SCC in milk, the bioluminescent assay based on determination of ATP from somatic cells ([ATPsom]) in milk was proposed earlier. However, this assay is still not widely used in practice owing to lower reliability compared with conventional methods such as direct microscopy and flow cytometry. We revised the bioluminescent SCC assay and developed a simple protocol based on determination of the total non-bacterial ATP concentration in milk. It was shown that the novel ATP-releasing agent Neonol-10 (oxy-ethylated iso-nonyl phenol) has superior performance providing 100% lysis of somatic cells while not disrupting bacterial cells of milk at a concentration of 1·5% w/w. There was high correlation (R2=0·99) between measured bioluminescence and SCC as measured by direct microscopy. The observed detection limit of the bioluminescent milk SCC assay was as low as 900 cell/ml, time of analysis was 2–3 min per sample. The proposed method has high potential for on-site mastitis diagnostics.

Warm vs Cold Sampling of Raw Milk for Direct Microscopic Somatic Cell Counting

1990 ◽  
Vol 53 (12) ◽  
pp. 1073-1075
Author(s):  
ROY GINN ◽  
VERNAL PACKARD
Keyword(s):  
Somatic Cell ◽  
Dairy Products ◽  
Somatic Cells ◽  
Raw Milk ◽  
Cell Counting ◽  
Microscopic Method ◽  
Grand Average ◽  
Cell Counts ◽  
Four Levels ◽  
Different Levels

Standard Methods for the Examination of Dairy Products (1) requires that milk be sampled cold for analysis of both bacteria and somatic cells by the direct microscopic method. Preliminary observations made at Dairy Quality Control Institute, Inc. indicated that better precision in somatic cell counting could be obtained if milk was sampled at 38°C rather than 0–4.4°C. A study was undertaken to confirm preliminary findings. Three technicians made direct microscopic somatic cell counts (DMSCC) in triplicate on milk samples standardized to four different levels of counts. The grand average counts at the four levels of cells for cold vs warm milk sampling were, in thousands of cells per ml, 149 vs 154, 410 vs 416, 705 vs 697, and 1034 vs 1024. Grand average standard deviations for cold vs warm, respectively, at the four levels of count were 19 vs 17, 47 vs 42, 54 vs 50, and 69 vs 62. Thus, an improvement in precision of counting was observed at all four levels of somatic cells when raw milk is warmed to 38°C prior to sampling.

Determination of Bacterial ATP Levels in Raw Milk: Selectivity of Non-Bacterial ATP Hydrolysis

1986 ◽  
Vol 49 (1) ◽  
pp. 4-7 ◽  
Author(s):  
DANIEL P. THERON ◽  
BERNARD A. PRIOR ◽  
PIETER M. LATEGAN
Keyword(s):  
Somatic Cell ◽  
Atp Hydrolysis ◽  
Somatic Cells ◽  
Enterobacter Cloacae ◽  
Raw Milk ◽  
Atp Content ◽  
Subsequent Determination ◽  
Limited Application

The selective destruction of non-bacterial ATP and subsequent determination of bacterial ATP using the ATP bioluminescent technique was investigated. Treatments to release ATP from somatic cells and hydrolyze free ATP also significantly reduced the ATP content of Enterobacter cloacae in skim and raw milk. The reduction can mainly be ascribed to apyrase (an ATPase) affecting the ATP content of intact bacteria. Somatic cell treatments failed to completely eliminate non-bacterial ATP. Although treatment with a somatic releasing reagent, EDTA and apyrase, resulted in a 96% reduction in the ATP content of raw milk, the remaining non-bacterial ATP was still considerably more than found in the bacterial component of raw milk studied here. Until reagents are available to selectively destroy all non-bacterial ATP without affecting the bacterial ATP content, the bioluminescent technique will have limited application in determination of the bacterial quality of raw milk.

scholarly journals Effect of somatic cell count on dairy products: a review

2017 ◽  
Vol 3 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Mukta Talukder ◽  
HM Manir Ahmed
Keyword(s):  
Somatic Cell ◽  
Cell Count ◽  
Somatic Cell Count ◽  
Proteolytic Enzymes ◽  
Somatic Cells ◽  
Raw Milk ◽  
Microbial Count ◽  
Endogenous Enzymes ◽  
Fluid Milk

Somatic cells are the most essential factors naturally present in milk, and somatic cell count (SCC) is used as an indicator of monitoring mastitis incidence in the herd and also to assess the quality of milk. In addition, SCC is frequently used to determine quality payments to dairy producers. The SCC is directly related to get maximum milk production from individual cow and a lower SCC indicates better animal health, as somatic cells originate only from inside the animal's udder. SCC monitoring is important because as the number of somatic cells increases, milk yield is likely to fall, primarily due to the damage to milk-producing tissue in the udder caused by mastitis pathogens and the toxins they produce, particularly when epithelial cells are lost. Keeping low SSC will allow good quality more raw milk and provide a better product to milk processors whether used as fluid milk or converted to milk based products. Somatic cells containing lipolytic and proteolytic enzymes lead to degrade major nutrients fats and proteins, respectively. Elevated SCC is related to udder inflammation, which leads to alter the normal microbial count and physicochemical parameters of milk, as well as the quality of heat treated fluid milk and milk based product. The objective of this review is to discuss on the SSC and endogenous enzymes released from somatic cells in raw milk as well as effect of somatic cells count and their endogenous enzymes in processed milk and milk based products.Asian J. Med. Biol. Res. March 2017, 3(1): 1-9

scholarly journals Prediction of total quarter milk somatic cell counts based on foremilk sampling

2009 ◽  
Vol 76 (3) ◽  
pp. 326-330 ◽  
Author(s):  
Olga Wellnitz ◽  
Marcus G Doherr ◽  
Marta Woloszyn ◽  
Rupert M Bruckmaier
Keyword(s):  
Dairy Cows ◽  
Somatic Cell ◽  
Cell Count ◽  
Somatic Cell Count ◽  
Practical Reasons ◽  
Udder Health ◽  
Somatic Cell Counts ◽  
Cell Numbers ◽  
Cell Counts

Determination of somatic cell count (SCC) is used worldwide in dairy practice to describe the hygienic status of the milk and the udder health of cows. When SCC is tested on a quarter level to detect single quarters with high SCC levels of cows for practical reasons, mostly foremilk samples after prestimulation (i.e. cleaning of the udder) are used. However, SCC is usually different in different milk fractions. Therefore, the goal of this study was the investigation of the use of foremilk samples for the estimation of total quarter SCC. A total of 378 milkings in 19 dairy cows were performed with a special milking device to drain quarter milk separately. Foremilk samples were taken after udder stimulation and before cluster attachment. SCC was measured in foremilk samples and in total quarter milk. Total quarter milk SCC could not be predicted precisely from foremilk SCC measurements. At relatively high foremilk SCC levels (>300×103 cells/ml) foremilk SCC were higher than total quarter milk. At around (50–300)×103 cells/ml foremilk and total quarter SCC did not differ considerably. Most interestingly, if foremilk SCC was lower than 50×103 cells/ml the total quarter SCC was higher than foremilk SCC. In addition, individual cows showed dramatic variations in foremilk SCC that were not very well related to total quarter milk SCC. In conclusion, foremilk samples are useful to detect high quarter milk SCC to recognize possibly infected quarters, only if precise cell counts are not required. However, foremilk samples can be deceptive if very low cell numbers are to be detected.

Optical Somatic Cell Counting and Total Protein Analysis in a Dairy Herd Improvement Program1

1977 ◽  
Vol 40 (10) ◽  
pp. 671-675 ◽  
Author(s):  
N. WANG ◽  
G. H. RICHARDSON
Keyword(s):  
Somatic Cell ◽  
Total Protein ◽  
Potassium Dichromate ◽  
Somatic Cells ◽  
Dairy Herd ◽  
Cell Counting ◽  
Ambient Conditions ◽  
Fresh Milk ◽  
Cell Counts ◽  
Cheese Industry

Milk sample preparation for Optical Somatic Cell Counter II operation was simplified by using a diluter to add fixative, mix, and dilute samples. Potassium dichromate preservative tablets produced a mean increase of 7,000 in somatic cell counts in fresh milk. Samples held at 20–23 C beyond 2 days or at 4–7 C beyond 4 days showed a reduction in somatic cell count. The mean somatic cells in 3 Holstein herds tested over a 6-month period was 3.8 × 105/ml. A 22-month survey of 52.6 thousand Utah Dairy Herd Improvement samples which were shipped under ambient conditions and then held at 5 C until tested, indicated 75% below 400,000 and 2.7% above 1.6 million somatic cells/ml. Casein, noncasein protein, total protein, fat and milk weight data were also obtained on the three herds. Multiple correlations were obtained. The best correlations suggested that testing for total protein and somatic cells in a central laboratory would estimate casein and noncasein protein. Such tests are most valuable for the cheese industry.

scholarly journals A Simple Protocol for the Determination of Lysostaphin Enzymatic Activity

Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 917
Author(s):  
Alexander V. Grishin ◽  
Svetlana V. Konstantinova ◽  
Irina V. Vasina ◽  
Nikita V. Shestak ◽  
Anna S. Karyagina ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Catalytic Efficiency ◽  
Bacterial Cells ◽  
Binding Domains ◽  
Simple Protocol ◽  
Specialized Equipment ◽  
Osmotic Lysis ◽  
Cross Bridges ◽  
Bacterial Peptidoglycan

Antibacterial lysins are enzymes that hydrolyze bacterial peptidoglycan, which results in the rapid death of bacterial cells due to osmotic lysis. Lysostaphin is one of the most potent and well-studied lysins active against important nosocomial pathogen Staphylococcus aureus. Similarly to most other lysins, lysostaphin is composed of enzymatic and peptidoglycan-binding domains, and both domains influence its antibacterial activity. It is thus desirable to be able to study the activity of both domains independently. Lysostaphin cleaves pentaglycine cross-bridges within the staphylococcal peptidoglycan. Here, we report the protocol to study the catalytic activity of lysostaphin on the isolated pentaglycine peptide that is based on the chromogenic reaction of peptide amino groups with ninhydrin. Unlike previously reported assays, this protocol does not require in-house chemical synthesis or specialized equipment and can be readily performed in most laboratories. We demonstrate the use of this protocol to study the effect of EDTA treatment on the lysostaphin enzymatic activity. We further used this protocol to determine the catalytic efficiency of lysostaphin on the isolated pentaglycine and compared it to the apparent catalytic efficiency on the whole staphylococcal cells. These results highlight the relative impact of enzymatic and peptidoglycan-binding domains of lysostaphin on its bacteriolytic activity.

THE EFFECT OF MIXING ON DISTRIBUTION OF SOMATIC CELLS IN BULK TANK MILK1

1971 ◽  
Vol 34 (10) ◽  
pp. 470-470 ◽  
Author(s):  
Gilbert E. Ward ◽  
David T. Berman
Keyword(s):  
Somatic Cell ◽  
Somatic Cells ◽  
Somatic Cell Counts ◽  
Cell Counts ◽  
Bulk Tank ◽  
Representative Samples

Agitation of milk in bulk tanks for at least 1 min is necessary so that representative samples for somatic cell counts can be obtained.

Electronic Somatic Cell Count—Chemical Method, DMSCC, and WMT Tests for Raw Milk

1976 ◽  
Vol 39 (12) ◽  
pp. 854-858 ◽  
Author(s):  
D. R. THOMPSON ◽  
V. S. PACKARD ◽  
R. E. GINN
Keyword(s):  
Somatic Cell ◽  
Cell Count ◽  
Confidence Intervals ◽  
Somatic Cell Count ◽  
Raw Milk ◽  
Field Method ◽  
Regression Equations ◽  
Somatic Cell Counts ◽  
Cell Counts ◽  
Coefficients Of Variation

The Direct Miscroscopic Somatic Cell Count — field method (DMSCC), Wisconsin Mastitis Test (WMT), and Electronic Somatic Cell Count (ESCC) were studied to determine variability and relationship to each other. The coefficients of variation computed at a DMSCC count near one million were 15.6% (DMSCC), 6.3% (WMT), and 4.2% (ESCC). Linear regression equations were determined for predicting DMSCC results by WMT and ESCC. The approximate width of the 95% confidence intervals for ESCC predicting DMSCC were ± 275,000 and for WMT predicting DMSCC were ± 600,000. The prediction of square root and log transformations of DMSCC by WMT exhibited narrower confidence intervals for low somatic cell counts, but wider intervals for high counts (greater than 1,000,000).

Comparison of the Automated with the Semi-Automatic Coulter Counter Method and the Direct Microscopic Somatic Cell Count (DMSCC) on Raw Milk Samples1

1979 ◽  
Vol 42 (7) ◽  
pp. 567-568 ◽  
Author(s):  
R. E. GINN ◽  
V. S. PACKARD ◽  
D. R. THOMPSON
Keyword(s):  
Somatic Cell ◽  
Cell Count ◽  
Somatic Cell Count ◽  
Particle Diameter ◽  
Raw Milk ◽  
Average Difference ◽  
Milk Samples ◽  
Cell Counts ◽  
Cell Counter ◽  
Electronic Cell Counter

The automatic Milk Cell Counter (MCC) and semi-automatic electronic cell counter (ESCC) of Coulter Electronics were compared with each other and with the direct microscopic cell count (DMSCC) on raw milk samples with various cell counts. The average DMSCC count on 241 samples of milk with Wisconsin Mastitis Test (WMT) results of 22 mm and higher was 55,000 cells/ml above the average MCC count when calibrated to a 4.4-μm minimum particle diameter. This difference is statistically significant at the 1% level. On 24 different raw milk samples of widely varying somatic cell count analyzed in replicate six times per sample, the standard deviations for replicate samples were 34,300, 34,900 and 136,000 for the MCC, ESCC and DMSCC, respectively. For these tests, the MCC had been calibrated to a 4.3-μm minimum particle diameter. The average difference between counts by the MCC and ESCC methods was only 6080/ml, but this was statistically significant at the 5% level. The average MCC count with the equipment set at 4.3-μm minimum particle diameter was 58,000 above the average DMSCC count.

The role of various milk fractions and the importance of somatic cells in the formation of germinant(s) for Bacillus cereus when milk is pasteurized

1977 ◽  
Vol 44 (3) ◽  
pp. 555-568 ◽  
Author(s):  
F. L. Davies
Keyword(s):  
Bacillus Cereus ◽  
Cell Count ◽  
High Speed ◽  
Serum Proteins ◽  
Somatic Cells ◽  
Skim Milk ◽  
Raw Milk ◽  
Cell Counts ◽  
Blood Serum Proteins ◽  
Fluff Layer

SummaryConfirmation was obtained for the occurrence of an interaction between high and low molecular weight fractions of milk during its pasteurization, resulting in the formation of germinant(s) for Bacillus cereus. Various milk fractions were added to supernatants or dialysates of raw skim-milk, pasteurized and assayed for germinant. Micellar casein obtained by high speed centrifugation of raw milk stimulated germinant formation, but whole casein obtained by acid precipitation was less stimulatory and individual casein fractions showed no stimulation. A membrane rich ‘fluff’ layer obtained by high speed centrifugation of raw milk was highly stimulatory and suggested the possibility that the somatic cell count of milk may be related to germinant production.Using milk from endotoxin infused and infected quarters of individual cows, a correlation between cell count and germinant formation was clearly demonstrated. Adjustment of the cell counts of individual milk samples was generally accompanied by corresponding changes in germinant formation, though removal of cells by filtration did not have this effect, possibly since germinant precursor had leached from the cells and remained in the milk. Pasteurization was necessary, not simply to express germinant from cells but to effect an interaction of germinant precursors. It is not clear whether the cell associated germinant precursor is derived from the cells themselves or from a related component. Addition of blood serum proteins was without stimulatory effect.Since low cell count milks sometimes supported appreciable germination after pasteurization, but showed still higher levels when the cell count was raised, germinant may be formed via 2 pasteurization dependent processes, one between milk constituents alone and the other involving both milk constituents and somatic cells.

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