p-Chloroamphetamine (PCA) suppresses ingestive behavior in male rats.

Ingestive behavior was activated in male rats by intraoral intake and intake from a bottle of 1-M solution of sucrose. Intraperitoneal injection of p-chloroamphetamine (PCA), releasing central 5-hydroxytryptamine (5-HT) from serotonergic nerve terminals, inhibited ingestion of the sucrose solution. Significant inhibition of sucrose intake by PCA was observed at 1.25 and 2.5 mg/kg dose in a bottle intake test, and at 5.0 mg/kg dose in an intraoral intake test. These findings suggested that 1.25 and 5.0 mg/kg of PCA suppressed appetitive ingestive behavior and consummatory ingestive behavior in male rats, respectively.     

The role of previous exposure in the appetitive and consummatory effects of orexigenic neuropeptides

The ingestion of foods is comprised of two distinct phases of behavior: appetitive and consummatory. While most food intake paradigms include both phases, the intraoral intake test emphasizes the stereotyped consummatory-phase by infusing a liquid food directly into the oral cavity. Several hypothalamic peptides have been shown to increase intake of chow in standard food intake paradigms and the current experiments sought to test whether these peptides would increase food intake in the intraoral intake paradigm. NPY, melanin-concentrating hormone (MCH) and orexin-A were infused into the third ventricle (i3vt) in a counterbalanced latin-square design just prior to rats getting 0.1M sucrose solution infused via indwelling intraoral catheters and compared it to intake on bottle tests with access to the same sucrose solution. On the first day, each peptide increased intraoral intake relative to saline in the between-subjects comparison. Moreover, intake of sucrose following i3vt saline increased as a function of training. By the final day of the experiment, rats receiving saline consumed as much sucrose as rats receiving NPY, MCH, or orexin-A. This finding was conceptually replicated in the second experiment in which rats drank sucrose freely from a bottle on the home cage. A third experiment directly assessed the role of previous exposure in the sucrose intake induced by NPY. Those results confirm that repeated exposure to sucrose increases baseline intake and attenuates the hyperphagic effect of NPY. These results are consistent with two conclusions: (1) NPY, MCH, and orexin-A increase both appetitive and consummatory-phase ingestive behaviors on initial exposures; (2) repeated training interacts with the effects of these orexigenic peptides.     

Type of negative feedback controlling sucrose ingestion depends on sucrose concentration

The sham intake of concentrated, but not weak, milk solutions requires up to three sham-feeding tests for intake to reach maximum (7). It is well known that the sham intake of concentrated (0.8 M) sucrose requires three or more sham-feeding tests to reach its maximum (4, 6, 17), but it is not known if this occurs with weaker sucrose solutions. We investigated this question by measuring the sham intake of seven concentrations of sucrose (0.025, 0.05, 0.1, 0. 2, 0.4, 0.6, and 0.8 M) during five sham-feeding tests. Sham intake of the three highest concentrations required up to three sham-feeding tests to reach maximum. Sham intake of the four lowest concentrations reached maximum in the first sham-feeding test. Our results show that the type of negative feedback that controls the intake of sucrose depends on its concentration. With weak solutions, intake is limited by a single direct, physiological, negative-feedback signal. When concentrated solutions are ingested, intake is controlled jointly by a direct physiological signal operating in conjunction with a labile one that loses its effectiveness with sham-feeding experience.     

Nectar feeding by the ant Camponotus mus: intake rate and crop filling as a function of sucrose concentration

In independent assays, workers of the ant Camponotus mus were conditioned to visit an arena where they found a large drop of sucrose solution of different concentrations, from 5 to 70% weight on weight (w/w). Single ants were allowed to collect the sucrose solution ad libitum, and feeding time, feeding interruptions, crop load, and intake rates were recorded. Feeding time increased exponentially with sucrose concentration, and this relationship was quantitatively described by the increase in viscosity with concentration corresponding to pure sucrose solutions. Ants collecting dilute solutions (5 to 15% w/w) returned to the nest with partial crop loads. Crop filling increased with increasing sucrose concentration, and reached a maximum at 42.6% w/w. Workers collecting highly concentrated solutions (70% w/w) also returned to the nest with a partially-filled crop, as observed for dilute solutions. Nectar intake rate was observed to increase with increasing sucrose concentration in the range 5 to 30% sucrose. It reached a maximum at 30.8%, and declined with increasing sucrose concentration. Results suggest that both sucrose concentration and viscosity of the ingested solution modulate feeding mechanics as well as the worker's decision about the load size to be collected before leaving the source.     

Sugar preferences, absorption efficiency and water influx in a Neotropical nectarivorous passerine, the Bananaquit (Coereba flaveola)

Nectarivory has evolved repeatedly in a number of unrelated bird taxa throughout the world and nectar feeding birds, regardless of their taxonomic affiliation, display convergent foraging and food processing adaptations that allow them to subsist on weak sugar solutions. However, phylogeny influences sugar type preferences of nectarivores. We investigated sugar preferences, assimilation efficiency and water flux in a Neotropical honeycreeper, the Bananaquit (Coereba flaveola; Coerebidae), a member of a radiation of tanagers and finches. Bananaquits showed no preference for nearly equicaloric (25% w/v) sucrose, glucose, fructose or glucose-fructose mixtures in pair-wise choice tests. In agreement with this lack of preference, they were equally efficient at absorbing sucrose and both hexoses. Apparent assimilation efficiency of these sugars was around 97.5%. In pair-wise tests, Bananaquits displayed a strong preference for the most concentrated sucrose solution when the lowest concentration ranged from 276 to 522 mM. Between 522 and 1120 mM sucrose solution concentrations, Bananaquits were able to adjust their volumetric food intake in order to maintain a constant energy intake. At solution concentration of 276 mM, birds could not maintain their rate of energy intake by increasing food consumption enough. We consider that at low sugar concentrations, Bananaquits faced a physiological constraint; they were unable to process food at a fast enough rate to meet their energy needs. We also explored the possibility that dilute nectars might be essential to sustain high water needs of Bananaquits by allowing them to control osmolarity of the food. Between 276 and 1120 mM sucrose solution concentrations, average amount of free water drunk by Bananaquits was independent of food concentration. They drank very little supplementary water and did not effectively dilute concentrated nectars. The evidence suggests that water bulk of dilute nectars is a burden to Bananaquits.     

Prior pregastric food stimulation and gastrin-releasing peptide1-27 (GRP) synergize to inhibit sham feeding.

The hypothesis that prior pregastric food stimulation is sufficient to reveal an inhibitory effect of gastrin-releasing peptide1-27 (GRP) on sham feeding was tested in 11 male rats equipped with chronic gastric cannulas. Rats were sham fed a high-carbohydrate solution during a 45-min test session, after 17-h food deprivation. GRP (16 or 32 microg/kg) or saline was injected intraperitoneally either at the onset or 5 or 15 min after the onset of sham feeding. This allowed for a 0-, 5-, or 15-min period of pregastric food stimulation before GRP or saline injections. Sham intake was recorded every 5 min, and behavior was observed every minute. GRP inhibited sham feeding when it was administered after 5 or 15 min of prior pregastric food stimulation, but not when it was administered at test onset. A nonsignificant increase in resting behavior and decrease in feeding behavior were associated with the decrease in sham feeding. No anomalous behaviors were noted. We conclude that a synergy between GRP and prior pregastric, presumably oral, food stimulation is sufficient to inhibit sham feeding.     

A moment-by-moment comparsion of sucrose and saccharin drinking by the rat

Comparisons were made between the ingestion patterns in ratsto a 0.2% sodium saccharin solution and to a 32% sucrose solutionin both short-term (30 min, one solution only) and long-term(23 h, solution versus water) tests. The resolution of measurementin the short- and long-term tests was 0.5 and 30 s respectively.Analysis programs for both procedures allowed for a quantificationof the ingestion patterns over time, showing details of thelick bursts in the short-term tests and ingestion bouts in the23-h tests. Although the quantities of sucrose and saccharinconsumed in the long-term tests were equal, the drinking patternsfor water, saccharin and sucrose were markedly different duringthe three testing periods, (i) There were fewer drinking boutsto the sucrose than to the saccharin or water, (ii) The averagebout of sucrose was much larger than the saccharin or waterbouts, (iii) The inter-bout intervals for sucrose were muchlonger than those for saccharin, (iv) Nearly half of the sucroseintake occurred during the ‘lights-on’ portion ofthe 23-h drinking period as compared to less than one-thirdfor saccharin or water, (v) Food intake when saccharin was presentwas equal to normal food intake when only water was available.However, in the presence of sucrose, the number and the sizeof feeding bouts decreased resulting in a 36% reduction in foodintake. Similar results were found in the short-term tests whencomparing sucrose and saccharin ingestion in that the quantitiesconsumed were not reliably different, but the ingestion patternswere, (i) The rats had many more bursts of licking saccharinthan sucrose, (ii) The saccharin bursts were much shorter thanthose for sucrose, (iii) Saccharin licking occurred off andon throughout the 30-min testing period while sucrose was consumedat a rapid rate at first and then terminated in 10–15min from the period onset. Inferences about the different tastesof saccharin and sucrose to the rat arc drawn from the detailedpattern analyses.     

Rapid calorie metering inad lib rats

Adult wistar rats of either sex housed in individual cages and fedad lib were used to investigate the effects of calorie content and taste on ingestion. A 15 min single-choice solution (water, quinine, sucrose, saccharin) test as well as 1 h mixed diet (stock diet, sucrose-mixed, saccharin-mixed, quinine-mixed) tests were used. Calorically-rich sucrose either in solution or in mixed-diet was preferentially ingested. Tasteper se has not influenced calorie intake, as intake on sweet saccharin (3.4 ±0.4 cal) or on bitter quinine (3.3 ±0.6 cal) was similar to intake on stock diet (4.8 ±0.8 cal). Increased 5 min intake on sucrose solution (4.6 ±0.1 ml) over intake of other test solutions (saccharin 3.5 ±0.2 ml, quinine, 1.2 ±0.3 ml, water 2.1 ±0.1 ml) and calorie intake suppression on 1 h stock diet immediately following sucrose solution intake, indicate rapid calorie metering, probably based on fast-acting specific gustatory signals     

Nectar feeding by the hovering hawk moth Macroglossum stellatarum: intake rate as a function of viscosity and concentration of sucrose solutions

Although nectar feeding in insects has long been studied, the knowledge of the effect of nectar energy content on the ingestion dynamics separately from the viscosity of the fluid is very limited. To determine the effects of both factors on the feeding behavior of the hovering hawk moth Macroglossum stellatarum, we developed a method to independently manipulate sucrose concentrations and viscosity. The intake rate was analyzed as a function of sucrose concentration, the concentration at constant viscosity (kept constant by adding tylose, an inert polysaccharide), and of the different viscosities of a 30% weight/weight (w/w) sucrose solution (by adding different amounts of tylose). By increasing the concentration, and thus its viscosity, the solution intake rate (in microl s (-1)) decreased beyond a 20% w/w sucrose solution. For a 30% sucrose solution, the intake rate decreased with increasing viscosity. At constant viscosity, the solution intake rate decreased beyond a 30% w/w sucrose solution. However, if we considered the quantity of sucrose ingested per unit time (sucrose intake rate), the same fitted maximum was attained for both series in which the sucrose concentration changed (33.6% w/w). Results suggest that the gustatory input affects the dynamics of fluid ingestion separately from the viscosity.     

Simultaneous observation of ingestive and copulatory behavior of the male rat.

In a preliminary test male rats were allowed to ingest a 1 M solution of sucrose from a drinking spout. After daily intake of sucrose became stabilized, the males were given a sexually receptive or non-receptive female and the bottle filled with sucrose solution simultaneously. The ingestive and copulatory behavior was observed for 60 min under illumination by a red lamp. The data obtained from this study showed that the ingestive behavior of males was suppressed by the presence of sexually receptive females and, conversely, the sexual behavior of males was not affected by the presence of a bottle of sucrose. These results suggest that the presence of a sexual partner inhibits appetitive ingestive behavior, i.e., the responses used by male rats to obtain food.     

Effects of melanin-concentrating hormone on licking microstructure and brief-access taste responses

The effects of intracerebroventricular application of melanin-concentrating hormone (MCH) on licking for sucrose, quinine hydrochloride (QHCl), and water solutions were evaluated in two experiments. In experiment 1, rats received 90-min access to sucrose and water solutions after MCH or vehicle microinjection to the third ventricle (3V). MCH increased intake largely through increases in the rate of licking early in the meal and in the mean duration of lick bursts, suggesting an effect on gustatory evaluation. Therefore, in experiment 2, brief access tests were used with a series of sucrose and QHCl concentrations to behaviorally isolate the effects of intracerebroventricular MCH on gustatory evaluation. MCH uniformly increased licking for all sucrose solutions, water, and weak concentrations of QHCl; however, it had no effect on licking for the strongest concentrations of QHCl, which were generally avoided under control conditions. Thus MCH did not produce nonspecific increases in oromotor activity, nor did it change the perceived intensity of the tastants. We conclude that MCH enhanced the gain of responses to normally accepted stimuli at a phase of processing after initial gustatory detection and after the decision to accept or reject the taste stimulus. A comparison of 3V NPY and MCH effects on licking microstructure indicated that these two peptides increased intake via dichotomous behavioral processes; although NPY suppressed measures associated with inhibitory feedback from the gut, MCH appeared instead to enhance measures associated with hedonic taste evaluation.     

NPY-leptin: opposing effects on appetitive and consummatory ingestive behavior and sexual behavior

Many studies have indicated that neuropeptide Y (NPY) stimulates and leptin inhibits food intake. In line with this, intracerebroventricular injection of NPY (10 microg) stimulated and leptin (10 microg) inhibited intake of a sucrose solution when female rats were required to obtain the solution from a bottle. However, NPY inhibited and leptin stimulated intake if the solution was infused intraorally. Thus NPY stimulates the responses used to obtain food but inhibits those used to consume food, and leptin has the opposite effects. To test the specificity of these responses the sexual behavior of male rats was examined. NPY-treated males showed minor deficits in sexual behavior but chose to ingest a sucrose solution rather than copulate with a female if offered the choice. By contrast, leptin-treated males ingested little sucrose and displayed an increase in ejaculatory frequency if given the same choice. It is suggested that NPY is not merely an orexigenic peptide, but one that directs attention toward food. Similarly, leptin may not be an anorexic peptide, but one that diverts attention away from food toward alternate stimuli.     

Preference for vegetable oils in the two-bottle choice test in mice

We evaluated in mice their preference for different vegetable oils using the two-bottle choice test. Mice showed preference for all vegetable oils (at 1% concentration in emulsion) tested, including corn oil, canola oil, and mixed vegetable oil, compared to a control fluid without oil. Mice also significantly preferred highly palatable sucrose solutions (5-20%) in this test. One percent corn oil was selected to the same extent as were these sucrose solutions. Mixtures of 1% corn oil and several concentrations of sucrose were preferentially selected as compared to corn oil or sucrose alone. The concentration-response curves of oil-sucrose mixtures vs sucrose (0.5-20% concentrations) were similar to those of sucrose vs control solutions. These results suggest that 1% corn oil and sucrose at the concentrations tested did not affect each other's preference. Chronic intake of 20% sucrose solution, but not 100% corn oil, changed the preference for lower concentrations of each, and both induced remarkable increases in body weight of mice. In conclusion, we showed that mice preferred vegetable oils, and this preference compared favorably with highly palatable sucrose solutions and was not changed by chronic intake of corn oil.     

Sucrose hydrolysis does not limit food intake by Pallas's long-tongued bats

Nectarivorous bats include very dilute nectar in their natural diet, and recent work with Pallas's long-tongued bat Glossophaga soricina showed that sugar (energy) intake rate decreased at dilute sucrose solutions. However, chiropterophillous nectar is composed mainly of the hexoses glucose and fructose. Because bats fed hexose nectar would save the delay of hydrolyzing sucrose, we hypothesized that sugar intake rate should be higher on this diet than on sucrose nectar. We compared intake response in Pallas's long-tongued bats offered 1 : 1 glucose-fructose (hexose) and sucrose diets at 5%, 10%, 20%, 30%, and 40% (mass/volume) sugar solutions. We also tested the hypothesis that sucrose hydrolysis limits food intake in bats. Intake response was the same in bats fed both types of diet: sugar intake rate was lower in dilute solutions and then increased with sugar concentration. Similar intake responses in both diets indicate that sucrose hydrolysis alone does not limit food intake and support the idea that the burden of processing excess water in dilute solutions plays a major role.     

Hindbrain administration of NMDA receptor antagonist AP-5 increases food intake in the rat

Hindbrain administration of MK-801, a noncompetitive N-methyl-D-aspartate (NMDA) channel blocker, increases meal size, suggesting NMDA receptors in this location participate in control of food intake. However, dizocilpine (MK-801) reportedly antagonizes some non-NMDA ion channels. Therefore, to further assess hindbrain NMDA receptor participation in food intake control, we measured deprivation-induced intakes of 15% sucrose solution or rat chow after intraperitoneal injection of either saline vehicle or D(-)-2-amino-5-phosphonopentanoic acid (AP5), a competitive NMDA receptor antagonist, to the fourth ventricular, or nucleus of the solitary tract (NTS). Intraperitoneal injection of AP5 (0.05, 0.1, 1.0, 3.0, and 5.0 mg/kg) did not alter 30-min sucrose intake at any dose (10.7 +/- 0.4 ml, saline control) (11.0 +/- 0.8, 11.2 +/- 1.0, 11.2 +/- 1.0, 13.1 +/- 2.2, and 11.0 +/- 1.9 ml, AP5 doses, respectively). Fourth ventricular administration of both 0.2 mug (16.7 +/- 0.6 ml) and 0.4 mug (14.9 +/- 0.5 ml) but not 0.1 and 0.6 mug of AP5 significantly increased 60-min sucrose intake compared with saline (11.2 +/- 0.4 ml). Twenty-four hour chow intake also was increased compared with saline (AP5: 31.5 +/- 0.1 g vs. saline: 27.1 +/- 0.6 g). Furthermore, rats did not increase intake of 0.2% saccharin after fourth ventricular AP5 administration (AP5: 9.8 +/- 0.7 ml, vs. saline: 10.5 +/- 0.5 ml). Finally, NTS AP5 (20 ng/30 nl) significantly increased 30- (AP5: 17.2 +/- 0.7 ml vs. saline: 14.6 +/- 1.7 ml), and 60-min (AP5: 19.4 +/- 0.6 ml vs. saline: 15.5 +/- 1.4 ml) sucrose intake, as well as 24-h chow intake (AP5: 31.6 +/- 0.3 g vs. saline: 26.1 +/- 1.2 g). These results support the hypothesis that hindbrain NMDA receptors participate in control of food intake and suggest that this participation also may contribute to control of body weight over a 24-h period.     

Intracerebroventricular administration of MK-801 increases food intake through mechanisms independent of gastric emptying

Systemic or hindbrain administration of MK-801, a noncompetitive N-methyl-D-aspartate receptor antagonist, increases meal size. To examine whether MK-801 enhances intake by increasing gastric emptying, we administered MK-801 (2.0 microg/3.0 microl) into the fourth ventricle [intracerebroventricular (ICV)] and measured feeding and gastric emptying of 5-ml NaCl or 15% sucrose loads. In a parallel experiment, we examined food intake and gastric emptying following intraperitoneal (IP) injection of MK-801 (100 microg/kg). MK-801, either IP or ICV, increased 30-min sucrose intake compared with control (12.3 +/- 0.7 vs. 9.8 +/- 0.5 and 16.6 +/- 2.0 vs. 10.7 +/- 0.7 ml, for IP and ICV administration, respectively). Also, IP MK-801 increased 5-min gastric emptying of NaCl (4.13 +/- 0.1 ml emptied) and sucrose (3.11 +/- 0.1 ml emptied) compared with control (3.75 +/- 0.2 and 2.28 +/- 0.1 ml emptied for NaCl and sucrose loads, respectively). In contrast, ICV MK-801 did not alter NaCl emptying (3.82 +/- 0.1 ml emptied) compared with control (3.82 +/- 0.3 ml emptied) and actually reduced gastric emptying of sucrose (2.1 +/- 0.2 and 2.94 +/- 0.1 ml emptied, for MK and vehicle, respectively). These data confirm previous results that systemic as well as hindbrain injection of MK-801 increases food intake. However, because ICV MK-801 failed to increase gastric emptying, these results indicate that MK-801 increases food intake through mechanisms independent of altered gastric emptying.     

Intravenous administration of oxytocin in rats acutely decreases deprivation-induced chow intake,but it fails to affect consumption of palatable solutions

Despite its limited ability to cross the blood-brain barrier, peripherally administered oxytocin (OT) acutely decreases food intake, most likely via the brainstem and hypothalamic mechanisms. Studies performed to date have focused mainly on the effects of subcutaneous or intraperitoneal OT on the consumption of only solid calorie-dense diets (either standard or high-fat), whereas it is unknown whether, similarly to central OT, peripherally administered peptide reduces intake of calorie-dilute and non-caloric palatable solutions. In this project, we established that 0.1 μg/kg intravenous (IV) OT is the lowest anorexigenic dose, decreasing deprivation-induced standard chow intake by ca. 40% in rats and its effect does not stem from aversion. We then used this dose in paradigms in which effects of centrally acting OT ligands on consumption of palatable solutions had been previously reported. We found that IV OT did not change episodic intake of individually presented palatable solutions containing 10% sucrose, 0.1% saccharin, combined 10% sucrose-0.1% saccharin or 4.1%. Intralipid and it failed to affect daily scheduled consumption of a sucrose solution in non-deprived rats. In a two-bottle choice test, IV OT did not shift animals’ preference from sucrose to Intralipid. Finally, OT injected IV prior to the simultaneous presentation chow and a sucrose solution in food-deprived rats significantly decreased chow intake, whereas sugar water consumption remained unchanged. We conclude that IV OT reduces deprivation-induced chow intake without causing aversion, but the dose effective in decreasing energy-driven consumption of high-calorie food fails to affect consumption of palatable calorie-dilute solutions.     

Oxytocin knockout mice demonstrate enhanced intake of sweet and nonsweet carbohydrate solutions

Oxytocin knockout (OT KO) mice display enhanced intake of nutritive and nonnutritive sweet solutions (i.e., sucrose and saccharin) compared with wild-type (WT) mice of the same C57BL/6 background strain. The present study further investigated the differential behavioral response of OT KO and WT mice to sucrose solutions and also examined intake preferences of OT KO and WT mice for palatable but nonsweet isocaloric solutions of carbohydrate and fat. A progressive ratio operant licking procedure demonstrated that OT KO and WT mice display a similar motivational drive to consume 10% sucrose. A series of two-bottle intake tests revealed that OT KO mice consume significantly larger amounts of both sweet and nonsweet carbohydrate solutions (i.e., sucrose, Polycose, and cornstarch) compared with WT cohorts. Intake pattern analyses revealed that OT KO mice overconsume carbohydrate solutions by initiating more drinking bouts compared with WT mice; bout sizes did not differ between the genotypes. In contrast, OT KO and WT mice did not differ in their intake of Intralipid, a palatable soybean oil emulsion. These findings indicate that the absence of OT in mice does not affect their appetitive drive to consume palatable sucrose solutions. Instead, the absence of OT may increase daily intake of palatable sweet and nonsweet solutions of carbohydrate (but not fat) by selectively blunting or masking processes that contribute to postingestive satiety.     

Taste threshold determination and side-preference in captive cockatiels (Nymphicus hollandicus)

The taste thresholds of caged cockatiels (Nymphicus hollandicus) for aqueous solutions of sodium chloride, citric acid, and sucrose were studied using two-choice taste-preference tests. The effects of location on the threshold were tested by putting the flavored solution in either the preferred or non-preferred locations (i.e. sides of cages) and offering water in the opposite location. Four parameters (total consumption, consumption from preferred side, consumption from non-preferred side, and proportion of test solution consumed) were measured at the end of 3-day test periods. Experiments were repeated with increasing concentration of test flavors until intake variables were significantly (p<0.05) affected. The results showed that birds distinguished (p<0.05) between purified water and 0.16 mol l(-1) sodium chloride, 0.36 mol l(-1) sucrose, or pH 5.5 citric acid. The likelihood of detecting a taste threshold was greater for sodium chloride and citric acid when these solutions were placed on the preferred side. In contrast, sucrose sensitivity was greater when this solution was offered on the non-preferred side.     

THE ASSESSMENT OF THE BACTERIOLOGICAL CONDITION OF MILK BOTTLES

SUMMARY: A study of the relative values of a number of bacteriological tests for assessing the condition of milk bottles indicated that the colony count of the bottle rinse solution on yeastrel milk agar incubated for 4 days at 30°, combined with a clot-on-boiling test applied to 1 ml. of rinse in 9 ml. of sterile milk after incubation for 72 hr. at 19–20°, gave the most useful results.
The mean of the ratios of colony counts at 30° to those at 37° was 15·1, while it was as high as 22·9 for rinses with 37° of over 600 for an unsatisfactory bottle should be retained when the test is done at 30°. The thermoduric colony count of rinses of milk bottles, even when laboratory pasteurized in milk, did not provide any additional information to that given by the colony count at 30° made without pasteurization. A high proportion of the organisms in bottle rinses survived laboratory pasteurization in milk, the survival rate being highest in efficiently treated bottles.
The clot-on-boiling test gave results in general agreement with colony counts and served to indicate the potential influence of badly contaminated bottles on the keeping quality of milk placed in them. A substantial proportion of rinses with satisfactory colony counts reduced methylene blue within 48 hr. at 19–20°.
Colony counts at 37° were on the average much lower for bottles treated with steam than for bottles submitted to detergent treatment in various types of bottle washing machines. Treatment of bottles by steam or hypochlorite was more efficiently done on the farms than at the dairies.