Yeast cells counts

Brewing yeast cell count/viability/vitality methods... 

A 43-year-old male in the surgical ICU after exploratory laparotomy following a motor vehicle accident develops fever that is unresponsive to broad-spectrum antibacterial therapy (piperacillin-tazobactam 3.75 g every 6 hours, gentamicin 120 mg every 8 hours, and vancomycin 1 g every 12 hours). The patient has a central venous catheter and a Foley catheter. Blood cultures are negative at the time, but the patient has yeast growing in the sputum and urine. Laboratory studies reveal a white blood cell count of 11,300 cells/mm3 (11.3 x 109/L). 

The patient is started on fluconazole 400 mg/day, but 3 days later has persistent fever and develops hypotension and decreased urine output. Blood cultures reveal a germ tube-negative yeast growing in the blood. Laboratory studies revealed a white blood cell count of 12,300/mm3 (12x109/L), aspartate aminotransferase 68 IU/L (1.13 pKat/L), alanine aminotransferase 75 IU/L (1.25 pKat/L), alkaline phosphatase 168 IU/L (2.8 pKat/L), and normal bilirubin. Serum creatinine is 1.8 mg/dL (159 pmol/L). 

Figure 19.4 Experimental procedure for the assessment of phototoxicity of formulations using the yeast assay. Formulations are spread on agar previously seeded by yeast cells. Photocytotoxicity is assessed by colonies counting after growth on complete medium, whereas genetically altered colonies (here gene conversion involving the t ptophan locus) are detected using selective growth medium (here tryptophan-free), [39].

Batch culture experiments were performed to measure the rate of glucose consumption. Once the seed yeast had been incubated for exactly 48 h, the serum vials were each injected with 0.2 mL of freshly vortexed yeast. The moment of inoculation became time zero (f = 0) for the experiment. Viable cell counts taken at time zero showed the initial cell concentration to be on the order of 100,000 colony-forming units/mL. Samples were taken every 2 h over the next 12 h, except for the first 4 hours, and analyzed for glucose. 

We have found that turbidity measurements can be used as a routine test to keep track of the yeast cell population. Fermentations routinely were examined microscopically for cell count and viability. We use a 1% methylene blue stain to differentiate viable and dead yeast cells. The dead cells take the stain. 

Figure 8-31. Distribution of radioactive materials after paper chromatography of a pancreatin digest of denatured yeast cells which had incorporated S-sulfate from the medium. The chromatogram was run on Whatman 3MM paper and developed in n-butanol-acetic acid-water. Individual strips 0.5 cm long were counted in a liquid scintillation counter. Met, Met-O, O, and F indicate S-methionine, S-methionine sulfoxide, origin, and solvent front, respectively. [From R. Graham and W. M. Stanley, Anal. Biochem., 47 505 (1972).]...

Fig. 12.6. The onset of synthesis of various mitochondrial polypeptides upon transferring anaerobically grown yeast cells to aerobic conditions. Yeast cells were grown overnight under anaerobic conditions. At time zero they were transferred to aerobic conditions, and at the indicated time periods samples of cells were removed and lysed in the presence of NaOH and mercaptoethanol. Samples containing about 50 /ig of protein were electrophoresed in a sodium dodecyl sulfate-polyacrylamide gel. The proteins were electrotransferred to nitrocellulose sheets and decorated with specific antibodies and l-labelled protein A. Paper pieces corresponding to the labelled protein spots were cut out from the immune blot and counted in a y counter. The amount of counts obtained in the samples of 8 h aerobic conditions was taken as 100%. The antibodies used were directed against the following polypeptides porin of the mitochondrial outer membrane (29 k) /8 subunit of the proton-ATPase (iS-F,) subunit IV of cytochrome c oxidase (OxIV) and subunit V of cytochrome c oxidase (OxV).

The fermentative activity of the yeasts is almost the same in all industries. Differences in fermentation times arise mainly from differences in temperature. For the baking industry, temperature is high, therefore fermentation time is short, with Htde time for cell multipHcation and very high cell counts at the start. 

Ten microcuries of C -labeled inulin were added to 15.0 ml of a yeast suspension. The suspension was then centrifuged and the supernatant fluid carefully drawn off. The pellet of packed yeast occupied 0.2 ml and contained 10,000 CPM. The counting efficiency was 25%. Calculate the proportion of the packed yeast pellet that is interstitial space, assuming that the yeast cells were completely impermeable to the inulin and that the inulin did not adsorb to the cell surface. 

Under the given counting conditions (e.g., 0.2 ml of packed yeast cells Spread out and dried on a planchet, or suspended in a given volume of scintillation fluid), the efficiency of counring is 25%. 

FIGURE 1 Yeast cell growth, viability, sugar consumption, and ethanol production patterns of a typical fermentation. In a synthetic grape juice medium. Triple M (Spiropoulos et al., 2000) was used and inoculated with a commercial strain of 5. cerevisiae. Glucose and fructose concentrations were determined by enzymatic assay, viable cell counts by plating on YPD medium, and cell mass by absorbance at 580 nm. 

Figure 13.2-3. Yeast cell extract HPLC fractionation and LC-MS analysis. (A) Comparison of representative proteomic patterns recorded for two consecutive HPLC prefractionations. (B) Graphical comparison of HPLC prefractionation and straight analysis of the whole ceil extract (WCE) by LC-MS (a maximum protein spectral count was recorded for two successive HPLC runs and compared with the corresponding valne for the WCE). Black-to-red color shading indicates increasing spectral counts. (This fignre is available in full color at ftp //ftp.wiley.com/public/sci tech med/pharmaceutical biotech/.)...

Dubois medium is used for bacteria counts. Pimaricine (0.01%) is added to eliminate any yeast cells. 

There are several tests which are concerned with sedimentation rates of yeast samples. The Burns test [55] and its variants [56] measure the volume of yeast which sediments into the base of a conical centrifuge tube from a given volume of defined yeast suspension in a specified time. There have been modifications [57] in which the yeast is suspended in beer under carefully standardized conditions cell concentrations are measured over a number of hours, just below the surface of beer. Concentrations fall steadily until, at a time and cell count which is characteristic for the yeast, the rate of fall changes abruptly. Sedimentation then proceeds at a steady but much lower rate that is characteristic of single cells the faster initial rate is that of aggregated cells. Measurement of the increase in cell concentrations, following deflocculation with the enzyme pronase, has also been used [51]. 

Sample from brewery no. Total bacterial count (living and dead) per 10 yeast cells Viable cell count (per 10 yeast cells) ... 

It is important not to mix up vitality and viability of yeast cells. The vitality is the condition of the physiological capabilities of the cell, while viability describes if a cell is alive or dead. The viability is reported as a percentage of live cells (so live and dead cells are counted), whereas the vitality gives the status of the metabolic function (Report of Subcommittee, 2003 Van Zandycke, Simal, Gualdoni, Smart, 2003). 

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