Method for simulating fulminant sepsis caused by mixed infection with underlying burning injury. RU patent 2507601.
 Method and device for simulating infected oral mucosa wound in experiment / 2507600Infected oral mucosa wound is simulated on an internal surface of an animal's lower lip along the midline. That is ensured by applying original retractor clamps according to cl. 2 on the lower lip skin and mucosa. Branches of a Bilroth's hemostatic forceps are used to fix an oral mucosa area 2 mm2 to be turned about a longitudinal axis at an angle of 360°. The mucosa is detached towards the operator. The prepared wound is covered with a cultured bacterial suspension in the amount of 2 ml. The method is implemented with using a device that is the original retractor clamps comprising two plates placed on the lower lip skin and mucosa, coupled with a spring hinge and merging into retractor clamps levers. The plate placed onto the lower lip mucosa has a sharp edged hole of the diameter of 5 mm. The plate placed onto the lower lip skin has a knop having the diameter of 3.5 mm and the height of 2 mm. The inventions may be also used in experimental medicine to study the efficacy of new pharmaceutical preparations on the oral mucosal wound process in experimental animals. |
 Method for prevention of tumour development in experimental animals with high-frequency spontaneous tumour formation / 2507599Complex phytoadaptogenic preparation phytomix-40 (phm-40) in the form of 10% solution with drinking water is administered into CBA male mice suffering the high-frequency spontaneous hepatocarcinoma formation form the first month of the postnatal development, including a period to the completion of hepatic tissue differentiation. |
| Method for pharmacological stimulation of pedicle skin flap survival with nicorandyl / 2507598 Pedicle skin flap survival is corrected in laboratory animals is corrected by modelling a flap on the second experimental day. On the first and fourth day of the experiment, nicorandyl is introduced intraperitoneally in a daily dose of 1.3 mg/kg in two stages. |
| Method for pharmacological correction of skeletal muscle ischemia with nicorandyl / 2507597 Skeletal muscle ischemia in laboratory animals is corrected by simulating leg muscle ischemia on the second experimental day. That is ensured by the instragastric administration of nicorandyl 1.3 mg/kg in two stages on the first, fourth and seventh experimental day. |
| Method for pharmacological correction of skeletal muscle ischemia with l-norvaline / 2507596 Skeletal muscle ischemia in rats is corrected by simulating leg muscle ischemia on the second experimental day. The ischemia is corrected by the intragastric administration of L-norvaline in a daily dose of 10.0 mg/kg from the first experimental day every 46 hours for 7 experimental days. |
 Method for endothelial dysfunction correction with azithromycin in adma-like gestosis model in experiment / 2507595Method involves simulating gestosis in Wistar rats by the daily intraperitoneal introduction of L-nitro-arginine-methyl ester 25 mg/kg for 7 days. That is followed by the intragastric introduction of azithromycin 30 mg/kg once a day in the setting of the model above from 14th to 20th day of pregnancy. |
 Method for endothelial dysfunction correction in adma-like gestosis model in experiment / 2507594Method involves simulating gestosis in Wistar rats by the daily intraperitoneal introduction of L-nitro-arginine-methyl ester 25 mg/kg for 7 days from the 14th day of pregnancy. That is followed by a single simulation of a 10-minute remote ischemic episode on the hind limb on the 21st day of pregnancy by femoral artery clamping and reperfusion. Vascular tests follow 90 minutes later, and an endothelial dysfunction coefficient is calculated. |
 Method for simulating renoparenchymatous arterial hypertension in rats / 2507593Renoparenchymatous arterial hypertension is simulated in mature male rats by a chemical injury of the renal parenchyma by administering 4% paraformaldehyde 0.1 ml into an upper pole of both kidneys. |
 Method for detecting psychotropic activity of drug and drug-free substances / 2506649Invention refers to medicine, in particular to experimental pharmacology. Detecting the psychotropic properties of the analysed substances is ensured by modelling an emotional-physical stress situation reached by placing the animal into a cylinder with cold water. That is followed by recording a time for solving and fulfilling the task to escape from the cylinder with using the presented rescue aids (lath, stairs and cord) in the cylinder. A percentage probability of solving the task is calculated. Indices characterising the psychoemotional and locomotor effect of the analysed substances are calculated by specific formulas. |
 Method for simulating development of focal cerebral haemorrhage in brain cortex of newborn rats / 25058653-day newborn rats are placed in a chamber and exposed to the sound of intensity 70 dB, frequency 110 Hz for 60 minutes. |
 Method for modeling acute pancreatitis / 2244345As experimental animals one should apply mongrel dogs of 12-17 kg body weight. Under general anesthesia one should conduct superior-median laparotomy, introduce 3.0 ml 70%-ethanol solution under pancreatic capsule and then laparotomic wound should be sutured up. Manipulation should be performed once. The method provides modeling adequate acute pancreatic inflammation at no side effects being very simple in implementation. |
 Method and device for studying transosseous osteosynthesis model stiffness / 2246139Method involves studying transverse longitudinal and rotation stiffness characteristics. The studies are carried out step-by-step from the first order units to complete external fixation apparatus structure. The device has frame and is provided with calibration loads, wire rope, displacement indicators, strip for fastening to loading end of bone imitator fragment, beam for fixing displacement indicators, beams having unit for modeling longitudinal and transverse loadings. The frame is manufactured as parallelepiped. The fixing panel has openings for bone imitator, for fixing external fixation apparatus and yoke connection union and is fixed in end face part of the frame. Beam for fixing displacement indicators has longitudinal slit for fixing the indicators and arranging them on lateral slots in frame base. The beams having unit for modeling rotational, longitudinal and transverse loadings are arranged on lateral frame sides on lateral slots in base. |
 Method for experimental modeling urinary calculosis / 2248045The present innovation deals with modeling urinary calculosis in rats due to injecting intraperitoneally 60%-glucose solution at 1 ml/100 g animal body weight twice daily for 2 mo. The method is very simple and enables to achieve lithogenesis in 25% experimental animals. |
| Method for applying wave action to pathogenic microorganisms, viruses and tumor cells in experiment / 2250788 Method involves using Hann diode crystal with proper frequencies of pathogenic microorganisms and cells during their death period or during the stimulating factors action period being applied. |
 Method for modeling hypoxia with hypercapnia in animals / 2251158Hypoxia with hypercapnia should be modeled due to creating a closed system of inhaled air circulation. Air enters lungs out of hermetically sealed reservoir and at expiration returns back. The process of recirculation is supplied with an apparatus of artificial pulmonary ventilation. The innovation suggested provides steadiness in development of hypoxia with hypercapnia excluding the development of stressor reaction. Conditions should be created to carry out any manipulations with an animal in the course of an experiment. |
| Method for obtaining parodontitis model / 2252009 The present innovation should be carried out for the purpose to study ethiology and pathogenesis of parodontitis. One should affect with emotional stress in experimental animals (mature rats) due to placing 10-11 experimental animals into the cage at area of 0.018 sq. cm/animal. Before placing into the cage one should create artificial dental plaque around the cervix of the upper and lower incisors with the help of stomatological cement for every experimental animal. In the course of modeling all experimental animals should eat paste-like food. The method enables to shorten terms for obtaining the model desired and increase its similarity with pathomorphological manifestations of human parodontitis. |
 Acupuncture point electric model device / 2252743Device has input first variable resistor, capacitor and permanent resistor. Permanent resistor is connected to arm second and third variable resistors. Second ends of variable resistors are connected with motionless contacts of polarized relay. Movable contact of relay is connected to common bus. Input of device is connected to amplifier which has output connected with control wiring of polarized relay. Second end of wiring is connected with common bus. Device is intended for electrical modeling of balanced and misbalanced conditions of acupuncture point at electropunctural action with unlike-poled signals due to liquidation mutual errors at any circuit of opposite arms of the device. Values of active resistances can be installed independently at any arm of device. |
| Method for modeling hypoxic encephalopathy / 2253152 Laboratory animals should be once injected intraperitoneally or intravenously with phenylhydrazine at the dosage of 100-150 mg/kg. |
| Method for modeling chronic toxic nephropathy / 2253153 At studying the mechanisms of heavy metals toxic action, in particular, cadmium upon renal function, it is suggested to introduce cadmium sulfate solution into stomach once daily for 2 mo at the dosage of 0.5 mg/kg, on conversion to metal, where cadmium corresponds to 0.5 mg per 1 ml solution. The present innovation enables to study the pathology in dynamics of development and elaborate and searching preparations for treating and preventing chronic toxic nephropathy. |
| Method for stopping carcinoma cells division in a biological object / 2253903 Method involves exposing cell or cell group to external power source. At least two electrodes are introduced before treating the cells. One of electrodes is set on cytoplasmatic external cell membrane surface and the other one cell membrane and membrane potential value is measured. External electric voltage source is connected to the introduced electrodes oppositely in polarity with cell membrane potential difference value being not less than cell membrane potential. |
FIELD: medicine.
SUBSTANCE: fulminant sepsis is simulated by inducing a mixed infection with underlying a burning injury. For this purpose, a thermal injury of the area of 12-23% of the body surface in anaesthetised rabbits is applied by immersing the back and sides into water at temperature 90° for 10 seconds. That is followed by the subcutaneous introduction of the non-culturable culture Staphylococcus aureus 1.0 ml in the concentration of 105 microbial cells in 1 ml. Then 60 minutes later, the non-culturable culture Pseudomonas aeruginosa is subcutaneously introduced in the amount of 1.0 ml in the concentration of 105 microbial cells in 1 ml.
EFFECT: method enables studying the effect of the non-culturable mixed infection on the development of an infectious process caused in a susceptible organism, and developing the new laboratory diagnostic techniques; methods for treatment and prevention of fulminant sepsis in the environment of non-culturable bacteria circulation.
2 tbl, 2 ex
The invention relates to the experimental medicine and can be used for epidemiological investigation of outbreaks of infections associated with medical care, and study of pathogenetic mechanism of fulminant sepsis to search for new pathogenetic and etiotropic methods of treatment of sepsis.
The term «sepsis» Hippocrates suggested more than two thousand years ago, meaning this term decay of tissues, inevitably accompanied by decay, disease and death. Currently sepsis remains one of the most important problems of modern medicine into force progressively increasing trends in morbidity and high mortality rates [Krasnov M.V., V.M. Krasnov Sepsis in young children: modern criteria of diagnosis and treatment guidelines. / Clinical Microbiology and antimicrobial therapy// 2010, №1 (40). - P.28-39]. Sepsis is a frequent cause of mortality in the departments of reanimation and intensive therapy. Bacterial infection is most often the cause of septic shock. In the United States diagnosed annually 300-500 thousand cases of sepsis, and despite the increasing possibilities of intensive therapy in patients with septic shock mortality reaches 60%. [Beloborodov V.B. Pressing questions of diagnostics and treatment of sepsis. http//old.consilium-medicum.com/media/consilium/mdex.shtmlj. According to this year Spiridonova, et al. in patients with deep burns mortality in sepsis is 57.1% [Spiridonova this year, Smirnov S. Century, Lazareva E.B., N.V. Evdokimov, Menshikov DU Features bacteraemia and septicaemia in patients with thermal trauma /Emergency medicine. - 2011, №1. www.criticaLru/emergency/hage.php?what=articles&chapter=20111.
The main causative agent of sepsis is Pseudomonas aeruginosa. Microbe abundantly medical equipment, circulates among staff and patients, causes up to 20% of nosocomial infections, a third of all lesions of the urinary tract in patients with urological and considered the cause of 20-25% suppurative surgical infections and primary gram of bacteriemic diseases; often occurs in patients with burns and diseases of the bladder and reservoirs of infection can be the most different objects (for example, raw vegetables, flowers) [Medical Microbiology /Ed. V.I. Pokrovsky, D.C. Pozdeev. - M: GEOTAR MEDICINE, 1998. - 1200 C.].
M.T. Abidov, et al. proposed method of modelling staphylococcal sepsis [patent RU №2058598 from 20.04.1996]. For modeling of sepsis author suggested to use the drug suppressing the bactericidal activity of neutrophilic granulocytes. The drug is used intravenously. Against the background of this drug was injected intravenously culture cultivated Staphylococcus aureus in high concentrations (10 8 degree)typically spent only in laboratory conditions. Such conditions of occurrence of sepsis is practically not observed in clinical practice. First of all, people do not receive drugs that reduce the bactericidal activity of blood serum and intravenous penetration of microbes in such concentrations (10 8 degree) practically is not observed in patients. The proposed experimental model can be used only for research. In addition, in practical health care more often sepsis causes .
Proposed the method of modeling sepsis [patent RU №2058599 from 20.04.1996,]. Method does not simulate fulminant sepsis. The designed model enables the 4 day install the clinical picture of the development of sepsis. For stimulation of occurrence of sepsis authors used , artificial introduction of which inhibits the functional-metabolic activity of neutrophils and macrophages. This drug does not enter and therefore the mechanism of occurrence of sepsis in the experiment differs from diseases that occur in natural conditions. Disadvantages of the proposed method are playing and use of inadequately large infective dose for rabbit (0,5 .10 9 microbial cells). Infection of patients in the hospital premises at a dose usually occurs.
Proposed method of modelling of fulminant sepsis in rats as a result of perforation cecum fatal 92% of cases [Lundblad R. et al. Granulocyte colony-stimulating factor improves myelopoiesis and host defense in fulminant intra-abdominal sepsis in rats //Shock. 1995 Jul; 4 (1): 68-73, Ref.]. Sepsis caused by various bacteria in the gastrointestinal tract. One of the drawbacks of the proposed method is that not installed etiology pathogens causing sepsis and, therefore, cannot be etiotropic treatment of patients. In addition, the proposed method only be used for the development of methods of treatment of patients, not to identify in hospitals circulation of pathogens of infectious diseases, causing sepsis.
The closest technical solution chosen as a prototype, is a method of modelling of fulminant sepsis caused by Pseudomonas aeruginosa [Ridings, R. et al. Beneficial cardiopulmonary effects of pentoxifylline in experimental sepsis are lost once septic shock is established. // Arch Surg., 1994. - Nov; 129 (11): 1144-52, Ref.]. Disadvantages of the proposed method is: 1. The high concentration of Escherichia coli (5.10 8 microbial cells in 1 ml); 2. To play fulminant sepsis used monoinfection caused by Escherichia coli; 3. To play the experimental model used form of Pseudomonas aeruginosa. As is known, Pseudomonas aeruginosa refers to bacteria and is therefore more often is condition.
The present invention is a simple way to create experimental model of fulminant sepsis caused by in the conditions maximally close to natural infection of patients in hospitals.
The technical result is a simple, does not require large expenditures method based on the infection of laboratory animals bacteria Staphylococcus aureus and Pseudomonas aeruginosa while burn injuries. For the infection of animals used concentration of bacteria, which usually determined on the surface of the wounds in patients with burn disease.
The technical result is achieved by the fact that according to the invention rabbits applied thermal burn under anesthesia area 12-23% body surface area (by immersion), the back and side of the body in water at a temperature of 90° 10, then subcutaneously injected 1.0 ml culture Staphylococcus aureus in a concentration of 10 3 of microbial cells in 1 ml and after 60 minutes of entering the culture of Pseudomonas aeruginosa subcutaneously in a volume of 1.0 ml with a concentration of 10 5 microbial cells in 1 ml
At 1-3 days of the animals were observed for clinical signs of fulminant sepsis and death at the phenomena of septic shock.
The essence of the method is achieved by burning injury through the mechanism of stress and violation of immunological reactivity of the organism, and as a result of intoxication associated with tissue damage, causes a transition bacteria of P. aeruginosa in cultivated by the state with the release of enzymes aggression and increased permeability of fabrics. The result is a caused by Staphylococcus, Escherichia coli and intestinal sticks. Escherichia coli into the blood as a result of paresis of the small intestine and infects various organs and tissues of the body. The above factors cause the development of septic shock of the lightning.
The severity of endogenous intoxication in sepsis determined by the revenues into the bloodstream products of the decay of tissues. In violation of the barrier antitoxic function of the liver in blood serum increases the concentration of trace elements of variable valency, which include divalent iron. As activators of lipid peroxidation, Deplete reserves the antioxidant protection of the organism, which leads to hyperactivation of lipid peroxidation, ascending in systemic blood flow medium - and low-molecular peptides, the progression of endogenous intoxication and aggravation of sepsis and multiple organ failure and infectious-toxic shock.
The proposed method is as follows: rabbit put in a cage in accordance with the requirements of sanitary rules (Appr. Chief State sanitary doctor №1045-73) 3-day quarantine for adaptation rabbits to new cells in the experiment Animals are vivarium at air temperature 24-26 degC in accordance with the RF Ministry of health no. 267 dated 19.06.2003 and requirements of the European Convention (Strasbourg, 1986) on the content, feeding and caring for Guinea animals, their withdrawal from the experiment and the subsequent disposal. The rabbit removed the hair from the back and side of the trunk of 25% of the body surface.
Anesthesia was carried out according to the methodology proposed A.V. [Razin A.V. Influence of different variants of General anesthesia and surgical wound on the body of rabbits. Avtoref. kand. Diss.. Troitsk. - 2010]. Sedation spent 2% solution hydrochloride () in the dose of 5.0 mg/kg, and on its background intramuscularly injected with a 5% solution at a dose of 7.5 mg/kg body weight rabbit, respectively. In rabbits reported the condition of General anesthesia sufficient depth and duration to play experimental burn injury.
The surface of the back and side of the torso rabbit immersed in a water bath for 10 s at the temperature of 90°C. On the surface of burn imposed sterile and sterile gauze bandage and subcutaneous rabbit introduced 1.0 ml bacteria S. aureus in a concentration of 10 5 microbial cells in 1 ml, and then 60 min culture P.aeruginosa in a concentration of 1,0 ml 10 5 microbial cells in 1 ml bacteria received on the methods proposed .Б. Kozlov et al. [Patent RU №2470074 of 20.12.2012, a Way of extracting natural bacteria Staphylococcus]. Clinical manifestations registered within 21 days.
Rabbit breed «chinchilla» put it in a cage in accordance with the requirements of sanitary rules (Appr. Chief State sanitary doctor №1045-73) 3-day quarantine for adaptation of the rabbit to the new cage in the experiment. Rabbit contained in vivarium at air temperature 24-26 degC in accordance with the RF Ministry of health no. 267 dated 19.06.2003 and requirements of the European Convention (Strasbourg, 1986) on the content, feeding and caring for Guinea animals, their withdrawal from the experiment and the subsequent disposal. From the back and side of the body rabbit removed the hair.
Anesthesia was carried out according to the following procedure: Sedation spent 2% solution hydrochloride () in the dose of 5.0 mg/kg, and on its background intramuscularly injected with a 5% solution at a dose of 7.5 mg/kg body weight rabbit, respectively. Then immersed purified from wool surface in a water bath for 10 s at the temperature of 90°C. On the surface of burn imposed sterile and sterile gauze bandage and subcutaneous rabbit introduced 1.0 ml bacteria S. aureus in a concentration of 10 5 microbial cells in 1 ml and 60 min 1,0 ml culture of P. aeruginosa in a concentration of 10 3 of microbial cells in 1 ml of bacteria obtained by the method proposed .Б. Kozlov et al. [Patent RU №2470074 of 20.12.2012,]. In one day rabbit registered clinical symptoms of sepsis, and after 52 hours rabbit died as a result of caused by Escherichia coli and intestinal sticks and S. aureus. At the opening of the animal the size of a deep burn amounted to 13.0% of body surface area rabbit.
Using cultural methods to allocate S. aureus failed (sowing meat infusion, milk and salt, -salt and blood agar).
Pseudomonas aeruginosa stood out from all the organs of the stricken animal, and E. coli is allocated only from the heart. To highlight bacteria of P. aeruginosa conducted the sowing selective environment CPCH-agar, 5% blood agar and Endo agar. Identification of bacteria was carried out on the basis of the study of cultural and morphological properties of bacteria, unnecessary pigmentation, the ability to form , growth in the medium king And environment Hugh and agar, capacity growth at 42 degrees With and without growth at 5 degrees C.
E. coli allocated for the environment Endo at a temperature of 37 deg C. After 18-24 hours of incubation in thermostat with this environment selected red colony Escherichia and them on the glass with polyvalent OK serum containing antibodies to 22 enteropathogenic E. coli (). At positive agglutination reaction colony the notched agar and on the following day dedicated culture with polyvalent being investigated. At the final stage of serological identification put deployed agglutination reaction with specific serums. At positive deployed agglutination reaction allocated culture held a cultivation environment right bundle branch block for the study of and proteolytic properties.
Example 2
Rabbit breed «chinchilla» put it in a cage in accordance with the requirements of sanitary rules (Appr. Chief State sanitary doctor №1045-73) 3-day quarantine for adaptation of the rabbit to the new cage in the experiment. Rabbit contained in vivarium at air temperature 24-26 degC in accordance with the RF Ministry of health no. 267 dated 19.06.2003 and requirements of the European Convention (Strasbourg, 1986) on the content, feeding and caring for Guinea animals, their withdrawal from the experiment and the subsequent disposal.
From the back and side of the body rabbit removed the hair. Anesthesia was carried out according to the following procedure: Sedation spent 2% solution hydrochloride () in the dose of 5.0 mg/kg, and on its background intramuscularly injected with a 5% solution at a dose of 7.5 mg/kg body weight rabbit, respectively. Then immersed purified from wool surface rabbit in a water bath for 10 s at the temperature of 90°C. On the surface of burn imposed sterile and sterile gauze bandage and subcutaneous rabbit introduced 1.0 ml bacteria S.aureus in a concentration of 10 3 of microbial cells in 1 ml and 60 min 1,0 ml culture of P. aeruginosa in a concentration of 10 5 microbial cells in 1 ml of bacteria isolated by the technique proposed .Б. Kozlov et al. [Patent RU №2470074 of 20.12.2012,]. During the first day the rabbit registered clinical symptoms of sepsis and 17 hours rabbit died as a result of caused by P. aeruginosa and S. aureus. The burn area was 15,38% body surface area. culture of S. aureus and E. coli from the blood and internal organs allocate failed. Allocation and identification of Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli performed according to the methods described above.
A method of modelling of fulminant sepsis caused by bacteria of P. aeruginosa and S. aureus opens new opportunities for the investigation of epidemic outbreaks of infectious diseases associated with medical care in the etiology of which the leading role is played and bacteria and also allows research on testing effective pathogenetic and etiotrop treatment of patients with lightning sepsis occurred against the background of burn injury.
Table 2 item No.The hallmarks of the way
Prototype
The proposed method
1View of the microbes that cause sepsis
P. aeruginosa
S. aureus, P. aeruginosa, E. coli
2bacteria
and
3The period of clinical manifestations of sepsis (in hours)
528,7±2,8
4The number of the input dose of bacteria in 1 ml suspension
5.10 8 10 5 5Laboratory species
pigs Rabbits 6Scientific novelty of the invention
A patent describing the General level of development of the technology
Experience with bacteria opens a new direction in epidemiology, diagnosis, treatment and prevention of infectious diseases
Table 1Characteristics of the infectious process in rabbits caused by the application strains of S. aureus I. P. aeruginosa while burn injuries (n=14)
no CROs Lika
Body weight rabbit before the experiment (in grams)
Body weight rabbit at opening (in grams)
Size of a deep burn (in %)
The average daily loss of body weight (in grams)
Term mortality (in hours)
The allocation of organs rabbit at the opening of
P. aeruginosa
E. coli 1 2600 2500 12,66 100.0 39 + - 2 2300 2230 15,38 70,0 17 + - 4 3600 3525 17,77 75,0 18 + - 5 2100 2025 14,68 75,0 21 + - 6 2600 2500 22,85 100,0 49 + - 7 2560 2500 17,15 60,0 20 + - 8 2100 2045 16,66 55,0 18 + - 9 3150 3075 13,51 75,0 22 + - 10 3750 3660 17,77 90,0 36 + + 12 2700 2605 20,0 95,0 48 + + 13 3700 3630 12,45 70,0 22 + - 14 2600 2535 17,15 65,0 21 + - 15 2660 2600 16,58 60,0 19 + - 16 3300 3200 13,0 100,0 52 + +2837,14±129,92
2759,28±128,74
16, 32 pet±0,81
77,86±3,54
28,71±2,76
100% 21,4%Note: not included In the table rabbits under numbers 3 and 11 with an area of burn 14,1% and 14.7%, respectively, in connection with the that dogs have lived for 21 days.
A method of modelling of fulminant sepsis caused by while burn injuries wherein the rabbits applied thermal burn under anesthesia area 12-23% body surface area (by immersion), the back and side of the body in water at a temperature of 90° 10, then subcutaneously injected 1.0 ml culture Staphylococcus aureus in a concentration of 10 5 microbial cells in 1 ml and after 60 minutes of entering the culture of Pseudomonas aeruginosa subcutaneously in a volume of 1.0 ml with a concentration of 10 5 microbial cells in 1 ml